Sugarcane bacilliform viral (scbv) enhancer and its use in plant functional genomics

ABSTRACT

Identification of new enhancer sequence has significant utility in the plant functional genomics. The sugarcane bacilliform badnavirus (SCBV) transcriptional enhancer has been identified. This enhancer can be used to increase the rate of transcription from gene promoters and in activation tagging experiments. A ten-fold increase in transcription was observed when a 4× array of the SCBV enhancer was placed upstream of a truncated form of the maize alcohol dehydrogenase minimal promoter. Methods of using the SCBV transcriptional enhancer are described, as are chimeric transcription regulatory regions, constructs, cells, tissues, and organisms that comprise one or more copies of the enhancer.

PRIORITY CLAIM

This application is a continuation of co-pending U.S. application Ser.No. 13/220,564, filed Aug. 29, 2011, which claims priority to and thebenefit of U.S. Provisional Application No. 61/402,570, filed Aug. 30,2010. The entire disclosure of these prior applications, as well as thedisclosure of International Application No. PCT/US2011/049532, filedAug. 29, 2011, and published as WO 2012/030711, are incorporated hereinby reference in their entirety.

FIELD

The disclosure relates to the field of plant molecular biology andgenetic engineering, and specifically to polynucleotide molecules usefulfor modulating (e.g., enhancing) gene expression and/or proteinproduction in plants.

PARTIES TO JOINT RESEARCH AGREEMENT

This application describes and claims certain subject matter that wasdeveloped under a written joint research agreement between Agrigenetics,Inc., Mycogen Corporation, Exelixis Plant Sciences, Inc., and Exelixis,Inc., having an effective date of Sep. 4, 2007.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN ASCII TEXT FILE

A Sequence Listing is submitted herewith as an ASCII compliant text filenamed “Sequence_Listing.txt”, created on Jun. 10, 2014, and having asize of ˜1.6 kilobytes, as permitted under 37 CFR 1.821(c). The materialin the aforementioned file is hereby incorporated by reference in itsentirety.

BACKGROUND

There is an on-going need for genetic regulatory elements that direct,control or otherwise regulate expression of a transcribable nucleic acid(e.g., a transgene), for instance for use in a genetically engineeredorganism such as a plant. Genetic regulatory elements typically include5′ untranslated sequences such a transcription initiation regions thatcontain transcription factor and RNA polymerase binding site(s),enhancer/silencer elements, a TATA box and a CAAT box together with 3′polyadenylation sequences, transcription stop signals, translation startand stop signals, splice donor/acceptor sequences and the like.

For the purposes of genetic engineering, genetic regulatory elements aretypically included in an expression vector or other engineeredconstruct, to regulate expression of a transgene operably linked to theregulatory elements. Well known examples of promoters used in thisfashion are CaMV35S promoter (Nagy et al. In: Biotechnology in plantscience: relevance to agriculture in the eighties. Eds. Zaitlin et al.Academic Press, Orlando, 1985), maize ubiquitin promoter (Ubi;Christensen & Quail, Transgenic Research 5:213, 1996) and the Emupromoter (Last et al., Theor. Appl. Genet. 81 581, 1991), though manyothers will be known to those of ordinary skill. Likewise, enhancershave been isolated from various sources for use in genetic engineering;these include the cauliflower mosaic virus (35S CaMV) enhancer, afigwort mosaic virus (FMV) enhancer, a peanut chlorotic streakcaulimovirus (PC1SV) enhancer, or mirabilis mosaic virus (MMV) enhancer.

There is an on-going need to identify genetic regulatory elements, suchas enhancer domains, that can be harnessed to control expression ofsequences operably linked thereto, for instance in heterologous nucleicacid molecules such as vectors and other engineered constructs.

SUMMARY OF THE DISCLOSURE

The present disclosure describes novel transcription regulatory regionscomprising an enhancer domain and, under the enhancing control of theenhancer domain, a transcription regulatory domain. The enhancer domaincomprises a plurality (e.g., two to four or more) of copies of a naturalbut previously unrecognized SCBV enhancer arranged in tandem. Thetranscription regulatory regions (promoters) of the present disclosureprovide enhanced transcription as compared to the promoter in theabsence of the enhancer domain. In one example, a chimeric transcriptionregulatory region is disclosed comprising one or more copies of the SCBVenhancer element shown in position 337 to position 618 of SEQ ID NO: 1;and operably linked thereto, a promoter comprising an RNA polymerasebinding site and a mRNA initiation site, wherein when a nucleotidesequence of interest is transcribed under regulatory control of thechimeric transcription regulatory region, the amount of transcriptionproduct is enhanced compared to the amount of transcription productobtained with the chimeric transcription regulatory region comprisingthe promoter and not comprising the SCBV enhancer sequence.

DNA constructs are also provided comprising a described transcriptionregulatory region and a DNA sequence to be transcribed. In one example,a DNA construct comprises a disclosed transcriptional initiation regionoperably linked to a transcribable polynucleotide molecule operablylinked to a 3′ transcription termination polynucleotide molecule. TheDNA constructs provide for enhanced transcription of the DNA sequence tobe transcribed. Transgenic plants, plant cells or tissue (such as adicotyledon or a monocotyledon plants, plant cells or tissue)transformed with the disclosed constructs are also disclosed. Alsoprovided is a plant seed, fruit, leaf, root, shoot, flower, cutting andother reproductive material useful in sexual or asexual propagation,progeny plants inclusive of F1 hybrids, male-sterile plants and allother plants and plant products derivable from the disclosed transgenicplant. Methods of producing the disclosed transgenic plants, plant cellsor tissue are also provided herein.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequence of the SCBV promoter (corresponding topositions 6758-7596 of GenBank Accession No. AJ277091.1, “Sugarcanebacilliform IM virus complete genome, isolate Ireng Maleng” whichincorporated by reference herein in its entirety as it appeared on-lineon Apr. 15, 2010); this sequence is also shown in SEQ ID NO: 1. Theenhancer sequences defined in this study extend from −222 to −503 andare underlined in the Figure (corresponding to position 337 to position618 of SEQ ID NO: 1).

FIGS. 2A and 2B illustrate results of the analysis of the SCBV promoter.FIG. 2A shows fragments of the SCBV promoter containing sequences from−839 bp, −576 bp and −333 bp upstream of the transcription start siteand 106 bp downstream of the transcription start site fused to theluciferase (LUC) reporter gene. FIG. 2B shows a histogram of the ratioof LUC/GUS activity from HiII cells co-transformed with the plasmidsabove and an UBI::GUS reporter construct. The results show that thepromoter fragment containing sequences from −576 bp upstream of thetranscription start site had 60% of the activity of the promoterfragment containing 839 bp upstream of the start site. In contrast, thepromoter fragment containing sequences from −333 bp upstream of thestart site had only 10% of the activity of the full-length promoter(from −839 bp upstream of the transcription start site). Thus, sequencesinvolved in promoter activity reside upstream of the −333 bp.

FIGS. 3A and 3B illustrate that the SCBV enhancer elements describedherein enhance transcription from the maize Adh1 promoter. One, two andfour copies of the SCBV promoter sequences from −503 to −222 were clonedupstream of a truncated maize Adh1 promoter, fused to the fireflyluciferase gene (FIG. 3A). For comparison, 4 copies of the MMV enhancersequences and 2 copies of the MMV enhancer and 2 copies of the SCBVpromoter were cloned upstream of the truncated maize Adh1 promoter andfused to the firefly luciferase gene (FIG. 3A). These constructs werebombarded into maize Hi-II suspension cells along with the UBI::GUSreporter construct. As shown in FIG. 3B., constructs containing 1, 2 and4 copies of the SCBV enhancer had more than 5 times, 6 times and 10times more activity, respectively, than did cells bombarded with thetruncated Adh1 construct without any enhancers. The 4×MMV construct had2.5 times the activity as the truncated Adh1 construct and the 2×MMV2×SCBV construct had 6 times the activity as the truncated Adh1construct.

FIG. 4 shows accumulation of transcripts close to (“Flanking gene”) theintegration site of 4×SCBV in transgenic (T) plants comparednon-transgenic (W) control plants, analyzed using reverse transcriptionand PCR (RT-PCR). The level of housekeeping gene GAPDH is shown forcomparison. The 4×SCBV enhancer caused increased accumulation oftranscripts of genes near where it integrates; this increase intranscript accumulation probably results from an increased rate oftranscription.

SEQUENCE LISTING

The nucleic and/or amino acid sequences listed in the sequence listingbelow are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. Nucleic acid sequences (in the Sequence Listing orelsewhere herein) are presented in the standard 5′ to 3′ direction, andprotein sequences are presented in the standard amino (N) terminal tocarboxy (C) terminal direction.

SEQ ID NO: 1 shows the nucleic acid sequence of the SCBV promoter(corresponding to positions 6758-7596 of GenBank Accession No.AJ277091.1, “Sugarcane bacilliform IM virus complete genome, isolateIreng Maleng” incorporated by reference herein in its entirety as itappeared on-line on Apr. 15, 2010).

The enhancer elements described herein are from position 337 to position618 of SEQ ID NO: 1.

DETAILED DESCRIPTION I. Abbreviations

-   -   3′ UTR 3′-untranslated region    -   5′ UTR 5′-untranslated region    -   Adh1 alcohol dehydrogenase 1    -   asRNA antisense RNA    -   cDNA complementary DNA    -   dsRNA double-stranded RNA    -   GAPDH glyceraldehyde 3-phosphate dehydrogenase    -   KB kilobytes    -   kbp kilobase pairs    -   LUC luciferase    -   miRNA microRNA    -   nt nucleotide    -   ORF open reading frame    -   PCR polymerase chain reaction    -   RT-PCR reverse transcription and PCR    -   SCBV sugarcane bacilliform virus    -   siRNA small interfering RNA    -   ssRNA single stranded RNA    -   T_(m) thermal melting point    -   UTR untranslated region

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

5′ and/or 3′: Nucleic acid molecules (such as, DNA and RNA) are said tohave “5′ ends” and “3′ ends” because mononucleotides are reacted to makepolynucleotides in a manner such that the 5′ phosphate of onemononucleotide pentose ring is attached to the 3′ oxygen of its neighborin one direction via a phosphodiester linkage. Therefore, one end of apolynucleotide is referred to as the “5′ end” when its 5′ phosphate isnot linked to the 3′ oxygen of a mononucleotide pentose ring. The otherend of a polynucleotide is referred to as the “3′ end” when its 3′oxygen is not linked to a 5′ phosphate of another mononucleotide pentosering. Notwithstanding that a 5′ phosphate of one mononucleotide pentosering is attached to the 3′ oxygen of its neighbor, an internal nucleicacid sequence also may be said to have 5′ and 3′ ends.

In either a linear or circular nucleic acid molecule, discrete internalelements are referred to as being “upstream” or 5′ of the “downstream”or 3′ elements. With regard to DNA, this terminology reflects thattranscription proceeds in a 5′ to 3′ direction along a DNA strand.Promoter and enhancer elements, which direct transcription of a linkedgene, are generally located 5′ or upstream of the coding region.However, enhancer elements can exert their effect even when located 3′of the promoter element and the coding region. Transcription terminationand polyadenylation signals are located 3′ or downstream of the codingregion.

Agronomic trait: Characteristic of a plant, which characteristicsinclude, but are not limited to, plant morphology, physiology, growthand development, yield, nutritional enhancement, disease or pestresistance, or environmental or chemical tolerance are agronomic traits.An “enhanced agronomic trait” refers to a measurable improvement in anagronomic trait including, but not limited to, yield increase, includingincreased yield under non-stress conditions and increased yield underenvironmental stress conditions. Stress conditions may include, forexample, drought, shade, fungal disease, viral disease, bacterialdisease, insect infestation, nematode infestation, cold temperatureexposure, heat exposure, osmotic stress, reduced nitrogen nutrientavailability, reduced phosphorus nutrient availability and high plantdensity. “Yield” can be affected by many properties including withoutlimitation, plant height, pod number, pod position on the plant, numberof internodes, incidence of pod shatter, grain size, efficiency ofnodulation and nitrogen fixation, efficiency of nutrient assimilation,resistance to biotic and abiotic stress, carbon assimilation, plantarchitecture, resistance to lodging, percent seed germination, seedlingvigor, and juvenile traits. Yield can also affected by efficiency ofgermination (including germination in stressed conditions), growth rate(including growth rate in stressed conditions), ear number, seed numberper ear, seed size, composition of seed (starch, oil, protein) andcharacteristics of seed fill. Increased yield may result from improvedutilization of key biochemical compounds, such as nitrogen, phosphorousand carbohydrate, or from improved responses to environmental stresses,such as cold, heat, drought, salt, and attack by pests or pathogens.Recombinant DNA used in this disclosure can also be used to provideplants having improved growth and development, and ultimately increasedyield, as the result of modified expression of plant growth regulatorsor modification of cell cycle or photosynthesis pathways. Additionalexamples of agronomic traits, and altering such traits in plants, areprovided herein and/or will be recognized by those of ordinary skill inthe art.

Alterations: Alterations in a polynucleotide (for example, a polypeptideencoded by a nucleic acid of the present invention), as this term isused herein, comprise any deletions, insertions, and point mutations inthe polynucleotide sequence. Included within this definition arealterations to the genomic DNA sequence that encodes the polypeptide.Likewise, the term “alteration” may be used to refer to deletions,insertions, and other mutations in polypeptide sequences.

Altering level of production or expression: Changing, either byincreasing or decreasing, the level of production or expression of anucleic acid molecule or an amino acid molecule (for example an siRNA, amiRNA, an mRNA, a gene, a polypeptide, a peptide), as compared to acontrol level of production or expression.

Amplification: When used in reference to a nucleic acid, this refers totechniques that increase the number of copies of a nucleic acid moleculein a sample or specimen. An example of amplification is the polymerasechain reaction, in which a biological sample collected from a subject iscontacted with a pair of oligonucleotide primers, under conditions thatallow for the hybridization of the primers to nucleic acid template inthe sample. The primers are extended under suitable conditions,dissociated from the template, and then re-annealed, extended, anddissociated to amplify the number of copies of the nucleic acid. Theproduct of in vitro amplification can be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing, using standard techniques. Other examples of in vitroamplification techniques include strand displacement amplification (seeU.S. Pat. No. 5,744,311); transcription-free isothermal amplification(see U.S. Pat. No. 6,033,881); repair chain reaction amplification (seeWO 90/01069); ligase chain reaction amplification (see EP-A-320 308);gap filling ligase chain reaction amplification (see U.S. Pat. No.5,427,930); coupled ligase detection and PCR (see U.S. Pat. No.6,027,889); and NASBA™ RNA transcription-free amplification (see U.S.Pat. No. 6,025,134).

Antisense, Sense, and Antigene: DNA has two antiparallel strands, a5′→3′ strand, referred to as the plus strand, and a 3′→5′ strand,referred to as the minus strand. Because RNA polymerase adds nucleicacids in a 5′→3′ direction, the minus strand of the DNA serves as thetemplate for the RNA during transcription. Thus, an RNA transcript willhave a sequence complementary to the minus strand, and identical to theplus strand (except that U is substituted for T).

Antisense molecules are molecules that are specifically hybridizable orspecifically complementary to either RNA or the plus strand of DNA.Sense molecules are molecules that are specifically hybridizable orspecifically complementary to the minus strand of DNA. Antigenemolecules are either antisense or sense molecules directed to a DNAtarget. An antisense RNA (asRNA) is a molecule of RNA complementary to asense (encoding) nucleic acid molecule.

Antisense inhibition: This term refers to a class of gene regulationbased on cytoplasmic, nuclear, or organelle inhibition of geneexpression (e.g., expression for a host cell genome or the genome of apathogen, such as a virus) due to the presence in a cell of an RNAmolecule complementary to at least a portion of the mRNA beingtranslated.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA mayalso contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA is usuallysynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells or other samples.

Chimeric or Chimera: The product of the fusion of portions of two ormore different polynucleotide or polypeptide molecules. For instance,the phrases “chimeric sequence” and “chimeric gene” refer to nucleotidesequences derived from at least two heterologous parts. Chimericsequence may comprise DNA or RNA.

Chimeric transcription regulatory region: An array of nucleic acidcontrol or regulatory sequences that direct transcription of a nucleicacid operably linked thereto, which array is assembled from differentpolynucleotide sources. For instance, chimeric transcription regulatoryregions as described herein may be produced through manipulation ofknown promoters or other polynucleotide molecules. Chimerictranscription regulatory regions may combine one or more enhancerdomains with one or more promoters, for example, by fusing aheterologous enhancer domain from a first native promoter to a secondpromoter with its own partial or complete set of regulatory element(s).This disclosure provides, inter alia, chimeric transcription regulatoryregions that contain at least one SCBV enhancer domain fused (that is,operably linked) to a promoter active in plant(s).

Construct: Any recombinant polynucleotide molecule such as a plasmid,cosmid, virus, autonomously replicating polynucleotide molecule, phage,or linear or circular single-stranded or double-stranded DNA or RNApolynucleotide molecule, derived from any source, capable of genomicintegration or autonomous replication, comprising a polynucleotidemolecule where one or more transcribable polynucleotide molecule hasbeen operably linked.

Control plant: A plant that does not contain a recombinant DNA thatconfers (for instance) an enhanced or altered agronomic trait in atransgenic plant, is used as a baseline for comparison, for instance inorder to identify an enhanced or altered agronomic trait in thetransgenic plant. A suitable control plant may be a non-transgenic plantof the parental line used to generate a transgenic plant, or a plantthat at least is non-transgenic for the particular trait underexamination (that is, the control plant may have been engineered tocontain other heterologous sequences or recombinant DNA molecules).Thus, a control plant may in some cases be a transgenic plant line thatcomprises an empty vector or marker gene, but does not contain therecombinant DNA, or does not contain all of the recombinant DNAs, in thetest plant.

Cosuppression: The expression of a foreign (heterologous) gene that hassubstantial homology to an endogenous gene, resulting in suppression ofexpression of both the foreign and the endogenous gene.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached.

Triplets of nucleotides (referred to as codons) code for each amino acidin a polypeptide, or for a stop signal. The term codon is also used forthe corresponding (and complementary) sequences of three nucleotides inthe mRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule includes thereverse complement of that DNA molecule. Except wheresingle-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule.

Encode: A polynucleotide is said to encode a polypeptide if, in itsnative state or when manipulated by methods known to those skilled inthe art, the polynucleotide molecule can be transcribed and/ortranslated to produce a mRNA for and/or the polypeptide or a fragmentthereof. The anti-sense strand is the complement of such a nucleic acid,and the encoding sequence can be deduced therefrom.

Enhancer domain: A cis-acting transcriptional regulatory element (a.k.a.cis-element) that confers an aspect of the overall control of geneexpression. An enhancer domain may function to bind transcriptionfactors, which are trans-acting protein factors that regulatetranscription. Some enhancer domains bind more than one transcriptionfactor, and transcription factors may interact with different affinitieswith more than one enhancer domain. Enhancer domains can be identifiedby a number of techniques, including deletion analysis (deleting one ormore nucleotides from the 5′ end or internal to a promoter); DNA bindingprotein analysis using DNase I foot printing, methylation interference,electrophoresis mobility-shift assays, in vivo genomic foot printing byligation-mediated PCR, and other conventional assays; or by DNA sequencecomparison with known cis-element motifs using conventional DNA sequencecomparison methods. The fine structure of an enhancer domain can befurther studied by mutagenesis (or substitution) of one or morenucleotides or by other conventional methods. Enhancer domains can beobtained by chemical synthesis or by isolation from promoters thatinclude such elements, and they can be synthesized with additionalflanking nucleotides that contain useful restriction enzyme sites tofacilitate subsequence manipulation.

(Gene) Expression: Transcription of a DNA molecule into a transcribedRNA molecule. More generally, the processes by which a gene's codedinformation is converted into the structures present and operating inthe cell. Expressed genes include those that are transcribed into mRNAand then translated into protein and those that are transcribed into RNAbut not translated into protein (for example, siRNA, transfer RNA andribosomal RNA). Thus, expression of a target sequence, such as a gene ora promoter region of a gene, can result in the expression of an mRNA, aprotein, or both. The expression of the target sequence can be inhibitedor enhanced (decreased or increased). Gene expression may be describedas related to temporal, spatial, developmental, or morphologicalqualities as well as quantitative or qualitative indications.

Gene regulatory activity: The ability of a polynucleotide to affecttranscription or translation of an operably linked transcribable ortranslatable polynucleotide molecule. An isolated polynucleotidemolecule having gene regulatory activity may provide temporal or spatialexpression or modulate levels and rates of expression of the operablylinked transcribable polynucleotide molecule. An isolated polynucleotidemolecule having gene regulatory activity may include a promoter, intron,leader, or 3′ transcription termination region.

Gene Silencing: Gene silencing refers to lack of (or reduction of) geneexpression as a result of, though not limited to, effects at a genomic(DNA) level such as chromatin re-structuring, or at thepost-transcriptional level through effects on transcript stability ortranslation. Current evidence suggests that RNA interference (RNAi) is amajor process involved in transcriptional and posttranscriptional genesilencing.

Because RNAi exerts its effects at the transcriptional and/orpost-transcriptional level, it is believed that RNAi can be used tospecifically inhibit alternative transcripts from the same gene.

Heterologous: A type of sequence that is not normally (e.g., in thewild-type sequence) found adjacent to a second sequence. In oneembodiment, the sequence is from a different genetic source, such as avirus or organism or species, than the second sequence.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidconsists of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas base pairing. More specifically, A will hydrogen bond to T or U, andG will bond to C. In RNA molecules, G also will bond to U. Complementaryrefers to the base pairing that occurs between two distinct nucleic acidsequences or two distinct regions of the same nucleic acid sequence.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na concentration) of the hybridization bufferwill determine the stringency of hybridization. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed by Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11, hereinincorporated by reference.

The following is an exemplary set of hybridization conditions and is notmeant to be limiting.

Very High Stringency (Detects Sequences that Share 90% SequenceIdentity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Detects Sequences that Share 80% Sequence Identity orGreater)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Detects Sequences that Share Greater than 50% SequenceIdentity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

In cis: Indicates that two sequences are positioned on the same piece ofRNA or DNA.

In trans: Indicates that two sequences are positioned on differentpieces of RNA or DNA.

Industrial crop: Crops grown primarily for consumption by humans oranimals or for use in industrial processes (for example, as a source offatty acids for manufacturing or sugars for producing alcohol). It willbe understood that in many instances either the plant or a productproduced from the plant (for example, sweeteners, oil, flour, or meal)can be consumed; thus, a subset of industrial crops are food crops.Examples of food crops include, but are not limited to, corn, soybean,rice, wheat, oilseed rape, cotton, oats, barley, and potato plants.Other examples of industrial crops (including food crops) are listedherein.

Interfering with or inhibiting (expression of a target sequence): Thisphrase refers to the ability of a small RNA, such as a siRNA or a miRNA,or other molecule, to measurably reduce the expression and/or stabilityof molecules carrying the target sequence. A target sequence can includea DNA sequence, such as a gene or the promoter region of a gene, or anRNA sequence, such as an mRNA. “Interfering with or inhibiting”expression contemplates reduction of the end-product of the gene orsequence, e.g., the expression or function of the encoded protein or aprotein, nucleic acid, other biomolecule, or biological functioninfluenced by the target sequence, and thus includes reduction in theamount or longevity of the mRNA transcript or other target sequence. Insome embodiments, the small RNA or other molecule guides chromatinmodifications which inhibit the expression of a target sequence. It isunderstood that the phrase is relative, and does not require absoluteinhibition (suppression) of the sequence. Thus, in certain embodiments,interfering with or inhibiting expression of a target sequence requiresthat, following application of the small RNA or other molecule (such asa vector or other construct encoding one or more small RNAs), thesequence is expressed at least 5% less than prior to application, atleast 10% less, at least 15% less, at least 20% less, at least 25% less,or even more reduced. Thus, in some particular embodiments, applicationof a small RNA or other molecule reduces expression of the targetsequence by about 30%, about 40%, about 50%, about 60%, or more. Inspecific examples, where the small RNA or other molecule is particularlyeffective, expression is reduced by 70%, 80%, 85%, 90%, 95%, or evenmore.

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, e.g., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Metabolome: The complement of relatively low molecular weight molecules(metabolites) that is present in a single organism, a sample, a tissue,a cell, or whatever other division is divided. By way of example,metabolomes may include metabolic intermediates, hormones and othersignalling molecules, and secondary metabolites. Representativemetabolomes comprise the complement of metabolites found within abiological sample, such as a plant, plant part, or plant sample, or in asuspension or extract thereof. Examples of such molecules include, butare not limited to: acids and related compounds; mono-, di-, andtri-carboxylic acids (saturated, unsaturated, aliphatic and cyclic,aryl, alkaryl); aldo-acids, keto-acids; lactone forms; gibberellins;abscisic acid; alcohols, polyols, derivatives, and related compounds;ethyl alcohol, benzyl alcohol, methanol; propylene glycol, glycerol,phytol; inositol, furfuryl alcohol, menthol; aldehydes, ketones,quinones, derivatives, and related compounds; acetaldehyde,butyraldehyde, benzaldehyde, acrolein, furfural, glyoxal; acetone,butanone; anthraquinone; carbohydrates; mono-, di-, tri-saccharides;alkaloids, amines, and other bases; pyridines (including nicotinic acid,nicotinamide); pyrimidines (including cytidine, thymine); purines(including guanine, adenine, xanthines/hypoxanthines, kinetin);pyrroles; quinolines (including isoquinolines); morphinans, tropanes,cinchonans; nucleotides, oligonucleotides, derivatives, and relatedcompounds; guano sine, cytosine, adeno sine, thymidine, inosine; aminoacids, oligopeptides, derivatives, and related compounds; esters;phenols and related compounds; heterocyclic compounds and derivatives;pyrroles, tetrapyrroles (corrinoids and porphines/porphyrins, w/w/ometal-ion); flavonoids; indoles; lipids (including fatty acids andtriglycerides), derivatives, and related compounds; carotenoids,phytoene; and sterols, isoprenoids including terpenes.

MicroRNA (miRNA): Small, non-coding RNA gene products of approximately21 nucleotides long and found in diverse organisms, including animalsand plants. miRNAs structurally resemble siRNAs except that they arisefrom structured, foldback-forming precursor transcripts derived frommiRNA genes. Primary transcripts of miRNA genes form hairpin structuresthat are processed by the multidomain RNaseIII-like nuclease DICER andDROSHA (in animals) or DICER-LIKE1 (DCL1; in plants) to yield miRNAduplexes. The mature miRNA is incorporated into RISC complexes afterduplex unwinding. Plant miRNAs interact with their RNA targets withperfect or near perfect complementarity.

Nucleotide: The term nucleotide includes, but is not limited to, amonomer that includes a base linked to a sugar, such as a pyrimidine,purine or synthetic analogs thereof, or a base linked to an amino acid,as in a peptide nucleic acid (PNA). A nucleotide is one monomer in anoligonucleotide/polynucleotide. A nucleotide sequence refers to thesequence of bases in an oligonucleotide/polynucleotide.

The major nucleotides of DNA are deoxyadenosine 5′-triphosphate (dATP orA), deoxyguanosine 5′-triphosphate (dGTP or G), deoxycytidine5′-triphosphate (dCTP or C) and deoxythymidine 5′-triphosphate (dTTP orT). The major nucleotides of RNA are adenosine 5′-triphosphate (ATP orA), guanosine 5′-triphosphate (GTP or G), cytidine 5′-triphosphate (CTPor C) and uridine 5′-triphosphate (UTP or U). Inosine is also a basethat can be integrated into DNA or RNA in a nucleotide (dITP or ITP,respectively).

Oil-producing species (of plant): Plant species that produce and storetriacylglycerol in specific organs, primarily in seeds. Such speciesinclude but are not limited to soybean (Glycine nizax), rapeseed andcanola (such as Brassica napus, Brassica rapa and Brassica campestris),sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn (Zeamays), cocoa (Theobroina cacao), safflower (Carthamus tinctorius), oilpalm (Elaeis guineensis), coconut palm (Cocos nucifera), flax (Linumusitatissimuin), castor (Ricinus commiunis) and peanut (Arachishypogaea).

Oligonucleotide: An oligonucleotide is a plurality of nucleotides joinedby phosphodiester bonds, between about 6 and about 300 nucleotides inlength. An oligonucleotide analog refers to compounds that functionsimilarly to oligonucleotides but have non-naturally occurring portions.For example, oligonucleotide analogs can contain non-naturally occurringportions, such as altered sugar moieties or inter-sugar linkages, suchas a phosphorothioate oligodeoxynucleotide. Functional analogs ofnaturally occurring polynucleotides can bind to RNA or DNA

Operably linked: This term refers to a juxtaposition of components,particularly nucleotide sequences, such that the normal function of thecomponents can be performed. Thus, a first nucleic acid sequence isoperably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein-codingregions, in the same reading frame. A coding sequence that is “operablylinked” to regulatory sequence(s) refers to a configuration ofnucleotide sequences wherein the coding sequence can be expressed underthe regulatory control (e.g., transcriptional and/or translationalcontrol) of the regulatory sequences.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Percent sequence identity: The percentage of identical nucleotides in alinear polynucleotide sequence of a reference (“query”) polynucleotidemolecule (or its complementary strand) as compared to a test (“subject”)polynucleotide molecule (or its complementary strand) when the twosequences are optimally aligned (with appropriate nucleotide insertions,deletions, or gaps totaling less than 20 percent of the referencesequence over the window of comparison). Optimal alignment of sequencesfor aligning a comparison window are well known to those skilled in theart and may be conducted using tools such as the local homologyalgorithm of Smith and Waterman, the homology alignment algorithm ofNeedleman and Wunsch, the search for similarity method of Pearson andLipman. Such comparisons are preferably carried out using thecomputerized implementations of these algorithms, such as GAP, BESTFIT,FASTA, and TFASTA available as part of the GCG® Wisconsin Package®(Accelrys Inc., Burlington, Mass.). An “identity fraction” for alignedsegments of a test sequence and a reference sequence is the number ofidentical components which are shared by the two aligned sequencesdivided by the total number of components in the reference sequencesegment (that is, the entire reference sequence or a smaller definedpart of the reference sequence). Percent sequence identity isrepresented as the identity fraction multiplied by 100. The comparisonof one or more polynucleotide sequences may be to a full-lengthpolynucleotide sequence or a portion thereof, or to a longerpolynucleotide sequence. Substantial percent sequence identity is atleast about 80% sequence identity, at least about 90% sequence identity,or even greater sequence identity, such as about 98% or about 99%sequence identity.

Plant: Any plant and progeny thereof. The term also includes parts ofplants, including seed, cuttings, tubers, fruit, flowers, etc. Invarious embodiments, the term plant refers to cultivated plant species,such as corn, cotton, canola, sunflower, soybeans, sorghum, alfalfa,wheat, rice, plants producing fruits and vegetables, and turf andornamental plant species. The term plant cell, as used herein, refers tothe structural and physiological unit of plants, consisting of aprotoplast and the surrounding cell wall. The term plant organ, as usedherein, refers to a distinct and visibly differentiated part of a plant,such as root, stem, leaf or embryo.

More generally, the term plant tissue refers to any tissue of a plant inplanta or in culture. This term includes a whole plant, plant cell,plant organ, protoplast, cell culture, or any group of plant cellsorganized into a structural and functional unit.

Polynucleotide molecule: Single- or double-stranded DNA or RNA ofgenomic or synthetic origin; that is, a polymer of deoxyribonucleotideor ribonucleotide bases, respectively, read from the 5′ (upstream) endto the 3′ (downstream) end.

Polypeptide molecule: A polymer in which the monomers are amino acidresidues which are joined together through amide bonds. When the aminoacids are alpha-amino acids, either the L-optical isomer or theD-optical isomer can be used, the L-isomers being preferred. The termpolypeptide or protein as used herein encompasses any amino acidsequence and includes modified sequences such as glycoproteins. The termpolypeptide is specifically intended to cover naturally occurringproteins, as well as those that are recombinantly or syntheticallyproduced.

Post-Transcriptional Gene Silencing (PTGS): A form of gene silencing inwhich the inhibitory mechanism occurs after transcription. This canresult in either decreased steady-state level of a specific RNA targetor inhibition of translation (Tuschl, ChemBiochem, 2: 239-245, 2001). Inthe literature, the terms RNA interference (RNAi) andposttranscriptional cosuppression are often used to indicateposttranscriptional gene silencing.

Promoter: An array of nucleic acid control sequences which directtranscription of a nucleic acid, by recognition and binding of e.g., RNApolymerase II and other proteins (trans-acting transcription factors) toinitiate transcription. A promoter includes necessary nucleic acidsequences near the start site of transcription, such as, in the case ofa polymerase II type promoter, a TATA element. Minimally, a promotertypically includes at least an RNA polymerase binding site together andmay also include one or more transcription factor binding sites whichmodulate transcription in response to occupation by transcriptionfactors. Representative examples of promoters (and elements that can beassembled to produce a promoter) are described herein. Promoters may bedefined by their temporal, spatial, or developmental expression pattern.

A plant promoter is a native or non-native promoter that is functionalin plant cells.

Protein: A biological molecule, for example a polypeptide, expressed bya gene and comprised of amino acids.

Protoplast: An isolated plant cell without a cell wall, having thepotential for being transformed and/or regeneration into cell culture ora whole plant.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified fusionprotein preparation is one in which the fusion protein is more enrichedthan the protein is in its generative environment, for instance within acell or in a biochemical reaction chamber. Preferably, a preparation offusion protein is purified such that the fusion protein represents atleast 50% of the total protein content of the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Similarly, a recombinant protein is one encoded for by a recombinantnucleic acid molecule.

Regulatable promoter: A promoter the activity of which is regulated(directly or indirectly) by an agent, such as a transcription factor, achemical compound, an environmental condition, or a nucleic acidmolecule.

Regulating gene expression: Processes of controlling the expression of agene by increasing or decreasing the expression, production, or activityof an agent that affects gene expression. The agent can be a protein,such as a transcription factor, or a nucleic acid molecule, such as amiRNA or an siRNA molecule, which when in contact with the gene or itsupstream regulatory sequences, or a mRNA encoded by the gene, eitherincreases or decreases gene expression.

Regulatory sequences or elements: These terms refer generally to a classof polynucleotide molecules (such as DNA molecules, having DNAsequences) that influence or control transcription or translation of anoperably linked transcribable polynucleotide molecule, and therebyexpression of genes. Included in the term are promoters, enhancers,leaders, introns, locus control regions, boundary elements/insulators,silencers, Matrix attachment regions (also referred to as scaffoldattachment regions), repressor, transcriptional terminators (a.k.a.transcription termination regions), origins of replication, centromeres,and meiotic recombination hotspots. Promoters are sequences of DNA nearthe 5′ end of a gene that act as a binding site for RNA polymerase, andfrom which transcription is initiated. Enhancers are control elementsthat elevate the level of transcription from a promoter, usuallyindependently of the enhancer's orientation or distance from thepromoter. Locus control regions (LCRs) confer tissue-specific andtemporally regulated expression to genes to which they are linked. LCRsfunction independently of their position in relation to the gene, butare copy-number dependent. It is believed that they function to open thenucleosome structure, so other factors can bind to the DNA. LCRs mayalso affect replication timing and origin usage. Insulators (also knownas boundary elements) are DNA sequences that prevent the activation (orinactivation) of transcription of a gene, by blocking effects ofsurrounding chromatin. Silencers and repressors are control elementsthat suppress gene expression; they act on a gene independently of theirorientation or distance from the gene. Matrix attachment regions (MARs),also known as scaffold attachment regions, are sequences within DNA thatbind to the nuclear scaffold. They can affect transcription, possibly byseparating chromosomes into regulatory domains. It is believed that MARsmediate higher-order, looped structures within chromosomes.Transcriptional terminators are regions within the gene vicinity thatRNA polymerase is released from the template. Origins of replication areregions of the genome that, during DNA synthesis or replication phasesof cell division, begin the replication process of DNA. Meioticrecombination hotspots are regions of the genome that recombine morefrequently than the average during meiosis. Specific nucleotides withina regulatory region may serve multiple functions. For example, aspecific nucleotide may be part of a promoter and participate in thebinding of a transcriptional activator protein.

Isolated regulatory elements that function in cells (for instance, inplants or plant cells) are useful for modifying plant phenotypes, forinstance through genetic engineering.

RNA: A typically linear polymer of ribonucleic acid monomers, linked byphosphodiester bonds. Naturally occurring RNA molecules fall into threegeneral classes, messenger (mRNA, which encodes proteins), ribosomal(rRNA, components of ribosomes), and transfer (tRNA, moleculesresponsible for transferring amino acid monomers to the ribosome duringprotein synthesis). Messenger RNA includes heteronuclear (hnRNA) andmembrane-associated polysomal RNA (attached to the rough endoplasmicreticulum). Total RNA refers to a heterogeneous mixture of all types ofRNA molecules.

RNA interference (RNAi): Gene silencing mechanisms that involve smallRNAs (including miRNA and siRNA) are frequently referred to under thebroad term RNAi. Natural functions of RNAi include protection of thegenome against invasion by mobile genetic elements such as transposonsand viruses, and regulation of gene expression.

RNA interference results in the inactivation or suppression ofexpression of a gene within an organism. RNAi can be triggered by one oftwo general routes. First, it can be triggered by direct cellulardelivery of short-interfering RNAs (siRNAs, usually ˜21 nucleotides inlength and delivered in a dsRNA duplex form with two unpairednucleotides at each 3′ end), which have sequence complementarity to aRNA that is the target for suppression. Second, RNAi can be triggered byone of several methods in which siRNAs are formed in vivo from varioustypes of designed, expressed genes. These genes typically express RNAmolecules that form intra- or inter-molecular duplexes (dsRNA) which areprocessed by natural enzymes (DICER or DCL) to form siRNAs. In somecases, these genes express “hairpin”-forming RNA transcripts withperfect or near-perfect base-pairing; some of the imperfecthairpin-forming transcripts yield a special type of small RNA, termedmicroRNA (miRNA). In either general method, it is the siRNAs (or miRNAs)that function as “guide sequences” to direct an RNA-degrading enzyme(termed RISC) to cleave or silence the target RNA. In some cases, it isbeneficial to integrate an RNAi-inducing gene into the genome of atransgenic organism. An example would be a plant that is modified tosuppress a specific gene by an RNAi-inducing transgene. In most methodsthat are currently in practice, RNAi is triggered in transgenic plantsby transgenes that express a dsRNA (either intramolecular or hairpin, orintermolecular in which two transcripts anneal to form dsRNA).

RNA silencing: A general term that is used to indicate RNA-based genesilencing or RNAi.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences is expressed in terms of the similarity betweenthe sequences, otherwise referred to as sequence identity. Sequenceidentity is frequently measured in terms of percentage identity (orsimilarity or homology); the higher the percentage, the more similar thetwo sequences are. Homologs of the sequences referenced or disclosedherein, such as homologs of the SCBV enhancer element, will possess arelatively high degree of sequence identity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (Adv. Appl. Math. 2: 482, 1981); Needleman and Wunsch (J.Mol. Biol. 48: 443, 1970); Pearson and Lipman (PNAS USA 85: 2444, 1988);Higgins and Sharp (Gene, 73: 237-244, 1988); Higgins and Sharp (CABIOS5: 151-153, 1989); Corpet et al. (Nuc. Acids Res. 16: 10881-90, 1988);Huang et al. (Comp. Appls Biosci. 8: 155-65, 1992); and Pearson et al.(Methods in Molecular Biology 24: 307-31, 1994). Altschul et al. (NatureGenet., 6: 119-29, 1994) presents a detailed consideration of sequencealignment methods and homology calculations.

The alignment tools ALIGN (Myers and Miller, CABIOS 4: 11-17, 1989) orLFASTA (Pearson and Lipman, 1988) may be used to perform sequencecomparisons (Internet Program © 1996, W. R. Pearson and the Universityof Virginia, “fasta20u63” version 2.0u63, release date December 1996).ALIGN compares entire sequences against one another, while LFASTAcompares regions of local similarity. These alignment tools and theirrespective tutorials are available on the Internet atbiology.ncsa.uiuc.edu.

Orthologs or paralogs (more generally, homologs) of the disclosedsequences are typically characterized by possession of greater than 75%sequence identity counted over the full-length alignment with thesequence to which they are compared using ALIGN set to defaultparameters. Sequences with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least92%, at least 95%, or at least 98% sequence identity. In addition,sequence identity can be compared over the full length of one or bothbinding domains of the disclosed fusion proteins. In such an instance,percentage identities will be essentially similar to those discussed forfull-length sequence identity.

When significantly less than the entire sequence is being compared forsequence identity, homologs will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85%, at least 90%, at least 95%, or atleast 99% depending on their similarity to the reference sequence.Sequence identity over such short windows can be determined usingLFASTA; methods can be found at World Wide Web addressbiology.ncsa.uiuc.edu. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided. The present disclosureprovides not only the peptide homologs that are described above, butalso nucleic acid molecules that encode such homologs.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Conditions for nucleic acid hybridization andcalculation of stringencies can be found in Sambrook et al. (InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989)and Tijssen (Laboratory Techniques in Biochemistry and Molecular BiologyPart I, Ch. 2, Elsevier, New York, 1993). Nucleic acid molecules thathybridize under stringent conditions to the disclosed SCBV enhancersequences will typically hybridize to a probe based on either the entirefusion protein encoding sequence, an entire binding domain, or otherselected portions of the encoding sequence under wash conditions of0.2×SSC, 0.1% SDS at 65° C.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that each encode substantially the same protein.

Small interfering RNA (siRNA): RNA of approximately 21-25 nucleotidesthat is processed from a dsRNA by a DICER enzyme (in animals) or a DCLenzyme (in plants). The initial DICER or DCL products aredouble-stranded, in which the two strands are typically 21-25nucleotides in length and contain two unpaired bases at each 3′ end. Theindividual strands within the double stranded siRNA structure areseparated, and typically one of the siRNAs then are associated with amulti-subunit complex, the RNAi-induced silencing complex (RISC). Atypical function of the siRNA is to guide RISC to the target based onbase-pair complementarity.

Transcribable polynucleotide molecule: Any polynucleotide moleculecapable of being transcribed into a RNA molecule. Methods are known tothose of ordinary skill, for introducing constructs into a cell in sucha manner that the transcribable polynucleotide molecule is transcribedinto a functional mRNA molecule that is translated and thereforeexpressed as a protein product. Constructs may also be constructed to becapable of expressing antisense RNA molecules, in order to inhibittranslation of a specific RNA molecule of interest. Conventionalcompositions and methods for preparing and using constructs and hostcells are well known to one skilled in the art (see for example,Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3.Sambrook et al., Cold Spring Harbor Laboratory Press, 2000).

Transcription: The production of an RNA molecule by RNA polymerase as acomplementary copy of a DNA sequence.

Transcription termination region: Sequences that control formation ofthe 3′ end of a transcript. Self-cleaving ribozymes and polyadenylationsequences are examples of transcription termination sequences.

Transcriptional gene silencing (TGS): A phenomenon that is triggered bythe formation of dsRNA that is homologous with gene promoter regions andsometimes coding regions. TGS results in DNA and histone methylation andchromatin remodeling, thereby causing transcriptional inhibition ratherthan RNA degradation. Both TGS and PTGS depend on dsRNA, which iscleaved into small (21-25 nucleotides) interfering RNAs (Eckhardt, PlantCell, 14:1433-1436, 2002; Aufsatz et al., Proc. Natl. Acad. Sci. U.S.A.,99:16499-16506, 2002).

Transgenic: This term refers to a plant/fungus/cell/other entity ororganism that contains recombinant genetic material not normally foundin entities of this type/species (that is, heterologous geneticmaterial) and which has been introduced into the entity in question (orinto progenitors of the entity) by human manipulation. Thus, a plantthat is grown from a plant cell into which recombinant DNA is introducedby transformation (a transformed plant cell) is a transgenic plant, asare all offspring of that plant that contain the introduced transgene(whether produced sexually or asexually).

Transformation: Process by which exogenous DNA enters and changes arecipient cell. It may occur under natural conditions, or artificialconditions using various methods well known in the art. Transformationmay rely on any known method for the insertion of foreign nucleic acidsequences into a prokaryotic or eukaryotic host cell. Selection of themethod is influenced by the host cell being transformed and may include,but is not limited to, viral infection, electroporation, lipofection,and particle bombardment.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. Transformedcells include stably transformed cells in which the inserted DNA iscapable of replication either as an autonomously replicating plasmid oras part of the host chromosome. They also include cells that transientlyexpress the inserted DNA or RNA for limited periods of time. As usedherein, the term transformation encompasses all techniques by which anucleic acid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Transposon: A nucleotide sequence such as a DNA or RNA sequence that iscapable of transferring location or moving within a gene, a chromosomeor a genome.

Transgenic plant: A plant that contains a foreign (heterologous)nucleotide sequence inserted into either its nuclear genome ororganellar genome.

Transgene: A nucleic acid sequence that is inserted into a host cell orhost cells by a transformation technique.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector may also include one or more therapeuticgenes and/or selectable marker genes and other genetic elements known inthe art. A vector can transduce, transform or infect a cell, therebycausing the cell to express nucleic acids and/or proteins other thanthose native to the cell. A vector optionally includes materials to aidin achieving entry of the nucleic acid into the cell, such as a viralparticle, liposome, protein coating or the like.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Overview of Several Embodiments

The present disclosure describes novel transcription initiation regionscomprising an enhancer domain and, under the enhancing control of theenhancer domain, a transcription regulatory domain. The enhancer domaincomprises a plurality (e.g., two to four or more) of copies of a naturalbut previously unrecognized SCBV enhancer arranged in tandem. Thetranscription regulatory regions (promoters) of the present disclosureprovide enhanced transcription as compared to the promoter in theabsence of the enhancer domain. In one embodiment, a chimerictranscription regulatory region is disclosed comprising one or morecopies of the SCBV enhancer element shown in position 337 to position618 of SEQ ID NO: 1 (or a homolog thereof); and operably linked thereto,a promoter comprising an RNA polymerase binding site and a mRNAinitiation site, wherein when a nucleotide sequence of interest istranscribed under regulatory control of the chimeric transcriptionregulatory region, the amount of transcription product is enhancedcompared to the amount of transcription product obtained with thechimeric transcription regulatory region comprising the promoter and notcomprising the SCBV enhancer sequence(s). In some embodiments, thechimeric transcription regulatory region comprises a promoter obtainedfrom the upstream region of a plant virus gene, a bacterial gene, afungal gene, a plant nuclear gene, a plant extra-nuclear gene, aninvertebrate gene, or a vertebrate gene.

Also provided are DNA constructs comprising a described transcriptionregulatory region and a DNA sequence to be transcribed. In someembodiments, a DNA construct is disclosed comprising the transcriptionalinitiation region operably linked to a transcribable polynucleotidemolecule operably linked to a 3′ transcription terminationpolynucleotide molecule. In one embodiment, the transcribablepolynucleotide molecule confers an agronomic trait to a plant in whichit is expressed.

Also provided are transgenic plants. In one embodiment, a transgenicplant is stably transformed with a disclosed DNA construct. In someembodiments, the transgenic plant is a dicotyledon. In otherembodiments, the transgenic plant is a monocotyledon. In one particularembodiment, the transgenic plant is a maize plant.

Further provided is a seed of a disclosed transgenic plant. In oneembodiment, the seed comprises the disclosed DNA construct.

Even further provided is a transgenic plant cell or tissue. In oneembodiment, a transgenic plant cell or tissue comprises a disclosedchimeric transcription regulatory region. In some embodiments, the plantcell or tissue is derived from a dicotyledon. In other embodiments, theplant cell or tissue is from a monocotyledon. In one particularembodiment, the plant cell or tissue is from a maize plant.

Also provided are methods of producing a disclosed transgenic plant,plant cell, seed or tissue. In some embodiments, the method comprisestransforming a plant cell or tissue with a disclosed DNA construct.

Further provided are a plant cell, fruit, leaf, root, shoot, flower,seed, cutting and other reproductive material useful in sexual orasexual propagation, progeny plants inclusive of F1 hybrids,male-sterile plants and all other plants and plant products derivablefrom the disclosed transgenic plants.

Also disclosed is a maize plant cell, tissue or plant comprising one ormore copies of a SCBV enhancer element shown in position 337 to position618 of SEQ ID NO: 1. In one embodiment, a maize plant cell, tissue orplant comprises one or more copies of a SCBV enhancer element shown inposition 337 to position 618 of SEQ ID NO: 1 in which the one or morecopies of the SCBV enhancer element is inserted into a genome of themaize plant cell, tissue or plant at a random location. In someembodiments, the SCBV enhancer imparts enhanced transcription of anucleotide sequence of interest which is under regulatory control of theSCBV enhancer as compared to transcription of the nucleotide sequence ofinterest in the absence of the SCBV enhancer.

IV. SCBV Enhancer and its Uses

The present disclosure provides a previously unrecognized enhancerregion from the Sugarcane Bacilliform badnavirus (SCBV) genome, whichenhancer is useful in enhancing the transcription efficiency which mayresult in enhanced transcription of DNA sequences under control of theenhancer. Of particular interest is enhanced transcription of genesequences which may be of the same genetic origin as the host or offoreign origin, either the naturally occurring sequences (in both senseand antisense orientations) or synthetically prepared sequences. Thesubject enhancers comprise a plurality of two or more copies of apreviously unrecognized natural SCBV enhancer domain (the sequence ofwhich is provided in SEQ ID NO: 1, at positions 337 to 618). Theenhancer comprises at least two copies of the enhancer domain sequence,in some embodiments three or four or more copies, arranged in tandem.

Also contemplated are homologous enhancers. Without intending to belimited in any way, representative homologous sequences may includethose from other SCBV promoters, for instance from different SCBVisolates such as those described in Braithwaite et al. (Plant Cell Rep.23:319-326, 2004; incorporated herein by reference in its entirety) orin U.S. Pat. No. 5,994,123 (incorporated herein by reference in itsentirety).

A natural enhancer comprises a DNA sequence which in its nativeenvironment is upstream from and within about 600 bp of a promoter.Taking the initial nucleotide of the mRNA as 0, the sequence containingan enhancer is from about −50 to about −1,000 bp, usually from about −50to −950 bp, generally comprising about −100 to −800 bp. An enhancerdomain is cis-acting and desirably is located within about 10,000 bp,usually about 2,000 bp, more usually adjacent to or within about 1,000bp of a transcription initiation sequence to be enhanced. The enhancermay be in either orientation with respect to the transcriptioninitiation sequence and can be located upstream or downstream inrelation to the promoter it enhances, though it is usually upstream.

The enhancer domain of the present disclosure finds use with a widevariety of initiation sequences, including promoters that are naturallyfound under the control of the enhancer, e.g., in a cis position(adjacent and homologous) as well as those not normally associated withthe particular enhancer (e.g., heterologous). The enhancer domain andtranscription initiation domain may be from the same or differentkingdom, family or species. Species of interest include prokaryotes andeukaryotes, such as bacteria, plants, insects, mammals, etc.Combinations include the described SCBV (viral) enhancer domain(s) witha transcription initiation region of a structural gene of: a host forSCBV (e.g., from sugarcane), another plant species (e.g., of the same ora different family), an insect, a vertebrate animal, a bacterium, afungus, and so forth.

The disclosure also contemplates DNA constructs comprising a subjecttranscription initiation region and, under the control of thetranscription initiation region, a DNA sequence to be transcribed. TheDNA sequence may comprise a natural open reading frame includingtranscribed 5′ and 3′ flanking sequences. Alternatively, it may comprisean anti-sense sequence in that it encodes the complement of an RNAmolecule or portion thereof. When the construct includes an open readingframe (ORF) which encodes a protein, an enhanced transcriptioninitiation rate is obtained, usually providing an increased amount ofthe polypeptide expression product of the gene. When the constructcomprises an anti-sense sequence, the enhanced transcription of RNAcomplementary to wild type suppresses the expression of the wild typemRNA, thereby decreasing the amount of the polypeptide expressionproduct; it is contemplated that the wild type mRNA in question maycorrespond to a native mRNA of the host cell or a mRNA of a pathogen,such as a virus or fungus.

In various embodiments, the DNA sequence to be transcribed includes:protein encoding sequence(s) of a gene (e.g., from a plant, animal,bacterium, virus, or fungus), which may include: natural open readingframe(s) encoding a protein product; complementary DNA (cDNA) sequencesderived from mRNA encoded by a gene; synthetic DNA giving the desiredcoding sequence(s); protein encoding sequence(s) derived from exons of anatural gene, such as open reading frame(s) produced by exon ligation;and/or combinations of any two or more thereof. Attached to thesesequences are appropriate transcription termination/polyadenylationsequences; sequences from a natural gene (e.g., from a plant, animal,bacterium, virus, or fungus) that encodes a primary RNA product, that isconsisting of exons and introns (e.g., natural Polymerase II andPolymerase III transcribed genes of eukaryotes); synthetic DNA sequencesthat encode a specific RNA or protein product; sequences of DNA modifiedfrom a known coding sequence (e.g., a natural gene sequence) bymutagenesis (such as site specific mutagenesis) and/or other geneticengineering technology; chimeras of any of the above achieved byligation of DNA fragments, including chimeras that encode fusionproteins; and/or DNA sequences encoding the complement of RNA moleculesor portions thereof.

Enhanced transcription in plants may find use in enhancing theproduction of proteins characteristic of the plant (endogenous—that is,normally found in the wild-type host) or those proteins from othergenetic sources (exogenous—that is, not normally found in the wild-typehost). Examples of types of sequences to be expressed from the enhancersand chimeric transcription regulatory regions described herein include:antisense or small inhibitory RNAs (for gene suppression); nutritionallyimportant proteins; growth promoting factors; proteins giving protectionto the plant under certain environmental conditions, e.g., proteinsconferring resistance to metal, salt, or other toxicity; stress relatedproteins giving tolerance to extremes of temperature, freezing, etc.;proteins conferring pest or infection-related protection to the plant,e.g., proteins giving resistance to bacterial, fungal, or othermicrobial infection, or resistance to predation by insects (e.g., B.thuringiensis toxin) or to other invertebrate or vertebrate animals;compounds of medical importance outside of the plant, e.g.,anti-microbial, anti-tumor, etc.; proteins or other compounds ofspecific commercial value; increased level of proteins, e.g., enzymes ofmetabolic pathways (e.g., pathways for production of polyphenoliccompounds or other secondary metabolites); increased levels of productsof structural value to a plant host; and so forth. The sequences ofinterest which are transcribed will be of at least about 8 bp, at leastabout 12 bp, at least about 20 bp, and may be one or more kilobase pairs(kbp) in length.

V. Constructs

Constructs of the present disclosure typically contain a chimerictranscription regulatory region comprising one or more copies of theprovided SCBV enhancer element operably linked to a promoter (usuallycontaining at least an RNA polymerase binding site and a mRNA initiationsite), which region is operably linked to a transcribable polynucleotidemolecule operably linked to a 3′ transcription terminationpolynucleotide molecule. In addition, constructs may include but are notlimited to additional regulatory polynucleotide molecules from the3′-untranslated region (3′ UTR) of plant genes (e.g., a 3′ UTR toincrease mRNA stability of the mRNA, such as the PI-II terminationregion of potato or the octopine or nopaline synthase 3′ terminationregions). Constructs may include but are not limited to the5′-untranslated regions (5′ UTR) of an mRNA polynucleotide moleculewhich can play an important role in translation initiation and can alsobe a genetic component in a plant expression construct. For example,non-translated 5′ leader polynucleotide molecules derived from heatshock protein genes have been demonstrated to enhance gene expression inplants (see for example, U.S. Pat. Nos. 5,659,122 and 5,362,865 each ofwhich is incorporated by reference in its entirety). Such additionalupstream and downstream regulatory polynucleotide molecules as arepresent in the construct may be derived from a source that is native orheterologous with respect to the other elements present on theconstruct.

Thus, one embodiment is a construct comprising a chimeric transcriptionregulatory region itself comprising one or more copies (e.g., two,three, four or more copies) of the SCBV enhancer element shown inposition 337 to position 618 of SEQ ID NO: 1 (or a homolog thereof)operably linked to a promoter, operably linked to a transcribablepolynucleotide molecule so as to direct transcription of saidtranscribable polynucleotide molecule at a desired level and/or in adesired tissue or developmental pattern upon introduction of theconstruct into a plant cell. The transcribable polynucleotide moleculein some examples comprises a protein-coding region of a gene, and thechimeric transcription regulatory region provides transcription of afunctional mRNA molecule that is translated and expressed as a proteinproduct from the construct. In another embodiment, the transcribablepolynucleotide molecule comprises an antisense region of a gene, and thechimeric transcription regulatory region affects transcription of anantisense RNA molecule or other similar inhibitory RNA in order toinhibit expression of a specific RNA molecule of interest in a targethost cell.

Yet more example constructs of the present disclosure include double Tiplasmid border DNA constructs that have the right border (RB orAGRtu.RB) and left border (LB or AGRtu.LB) regions of the Ti plasmidisolated from Agrobacterium tumefaciens comprising a T-DNA, which alongwith transfer molecules provided by the Agrobacterium cells, enableintegration of the T-DNA into the genome of a plant cell. The constructsmay also contain plasmid backbone DNA segments that provide replicationfunction and antibiotic selection in bacterial cells, for example, anEscherichia coli origin of replication such as ori322, a broad hostrange origin of replication such as oriV or oriRi, and a coding regionfor a selectable marker such as Spec/Strp that encodes for Tn7aminoglycoside adenyltransferase (aadA) conferring resistance tospectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectablemarker gene. For plant transformation, representative host bacterialstrains include Agrobacterium tumefaciens ABI, C58, or LBA4404; however,other strains known to those skilled in the art of plant transformationcan be used.

Also contemplated are constructs comprising at least one SCBV enhancerelement (optionally in the context of a chimeric transcriptionregulatory region), which construct is an activation tagging construct.Activation tagging is a method by which genes are randomly and stronglyupregulated on a genome-wide scale, after which specific phenotypes canbe screened for and selected. Components useful in various types ofactivating tagging constructs are known; see, for instance: Walden etal., Plant Mol. Biol. 26: 1521-8, 1994 (describing an activation T-DNAtagging construct that was used to activate genes in tobacco cellculture allowing the cells to grow in the absence of plant growthhormones); Miklashevichs et al., Plant J. 12: 489-98, 1997; Harling etal., EMBO J. 16: 5855-66, 1997; Walden et al., EMBO J. 13: 4729-36, 1994(reports of genes isolated from plant genomic sequences flanking theT-DNA tag and putatively involved in plant growth hormone responses);Schell et al., Trends Plant Sci. 3: 130, 1998 (discussing investigationof a group of related studies); Kardailsky et al., Science 286:1962-1965, 1999 (describing activation T-DNA tagging and screening ofplants for an early flowering phenotype); Koncz et al., Proc Natl AcadSci USA 86(21):8467-71, 1989 (describing activation tagging using theAgrobacterium gene 5 promoter (pg5), which is active only inproliferating cells and must insert directly adjacent to a plant gene inorder to influence its expression); Wilson et al., Plant Cell 8:659-671, 1996 (activation tagging that utilizes a modified Ds transposoncarrying the CaMV 35S promoter and a nos::hpt selection cassette) andSchaffer et al., Cell 93: 1219-1229, 1998 (illustrating the same system,used to upregulate adjacent plant genes resulting in dominantgain-of-function mutations 1996); and Weigel et al., Plant Physiology,122:1003-1013, 2000 (illustrating activation tagging vectors that areuseful for screening tens of thousands of transformed plants formorphological phenotypes).

VI. Nucleotide Sequences for Transcription Enhancement

Exemplary transcribable polynucleotide molecules for transcriptionenhancement by incorporation into constructs as provided herein include,for example, polynucleotide molecules or genes from a species other thanthe target species or genes that originate with or are present in thesame species, but are incorporated into recipient cells by geneticengineering methods rather than classical reproduction or breedingtechniques. The type of polynucleotide molecule can include but is notlimited to a polynucleotide molecule that is already present in thetarget plant cell, a polynucleotide molecule from another plant, apolynucleotide molecule from a different organism, or a polynucleotidemolecule generated externally, such as a polynucleotide moleculecontaining an antisense message of a gene, or a polynucleotide moleculeencoding an artificial, synthetic, or otherwise modified version of atransgene.

In one embodiment, a polynucleotide molecule as shown in positions 337to 618 of SEQ ID NO: 1 (or two or more copies thereof) (for instance, inthe context of a chimeric transcription initiation region) isincorporated into a construct such that the described SCBV enhancersequence (or series of two or more such sequences) is operably linked toa transcribable polynucleotide molecule that is a gene of agronomicinterest or other expression sequence (more generally, a nucleotidesequence of interest). As used herein, the term “gene of agronomicinterest” refers to a transcribable polynucleotide molecule thatincludes but is not limited to a gene that provides a desirablecharacteristic associated with plant morphology, physiology, growth anddevelopment, yield, nutritional enhancement, disease or pest resistance,or environmental or chemical tolerance. The expression of a gene ofagronomic interest is desirable in order to confer an agronomicallyimportant trait, for instance. A gene of agronomic interest thatprovides a beneficial agronomic trait to crop plants may be, forexample, one or more sequences conferring to a plant expressing thegene: herbicide resistance (see, e.g., U.S. Pat. Nos. 6,803,501;6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425;5,633,435; 5,463,175; and U.S. Publications US20030135879 andUS20030115626), increased yield (see, e.g., U.S. Pat. RE38,446; U.S.Pat. Nos. 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828;6,399,330; 6,372,211; 6,235,971; 6,222,098; 5,716,837), insect control(see, e.g., U.S. Pat. Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046;6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655;6,538,109; 6,537,756; 6,521,442; 6,501,009; 6,468,523; 6,326,351;6,313,378; 6,284,949; 6,281,016; 6,248,536; 6,242,241; 6,221,649;6,177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695; 6,063,756;6,063,597; 6,023,013; 5,959,091; 5,942,664; 5,942,658, 5,880,275;5,763,245; 5,763,241), fungal disease resistance (see, e.g., U.S. Pat.Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671;5,773,696; 6,121,436; 6,316,407; 6,506,962), virus resistance (see,e.g., U.S. Pat. Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864;5,850,023; 5,304,730), nematode resistance (see, e.g., U.S. Pat. No.6,228,992), bacterial disease resistance (see, e.g., U.S. Pat. No.5,516,671), plant growth and development (see, e.g., U.S. Pat. Nos.6,723,897; 6,518,488), starch production (see, e.g., U.S. Pat. Nos.6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oilsproduction (see, e.g., U.S. Pat. Nos. 6,444,876; 6,426,447; 6,380,462),high oil production (see, e.g., U.S. Pat. Nos. 6,495,739; 5,608,149;6,483,008; 6,476,295), modified fatty acid content (see, e.g., U.S. Pat.Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538;6,589,767; 6,537,750; 6,489,461; 6,459,018), fiber production (see,e.g., U.S. Pat. Nos. 6,576,818; 6,271,443; 5,981,834; 5,869,720), highprotein production (see, e.g., U.S. Pat. No. 6,380,466), fruit ripening(see, e.g., U.S. Pat. No. 5,512,466), improved digestibility (see, e.g.,U.S. Pat. No. 6,531,648), improved flavor (see, e.g., U.S. Pat. No.6,011,199), low raffinose (see, e.g., U.S. Pat. No. 6,166,292), enhancedanimal and/or human nutrition (see, e.g., U.S. Pat. Nos. 6,723,837;6,653,530; 6,541,259; 5,985,605; 6,171,640), environmental stressresistance (see, e.g., U.S. Pat. No. 6,072,103), desirable peptides(e.g., pharmaceutical or secretable peptides) (see, e.g., U.S. Pat. Nos.6,812,379; 6,774,283; 6,140,075; 6,080,560), improved processing traits(see, e.g., U.S. Pat. No. 6,476,295), industrial enzyme production (see,e.g., U.S. Pat. No. 5,543,576), nitrogen fixation (see, e.g., U.S. Pat.No. 5,229,114), hybrid seed production (see, e.g., U.S. Pat. No.5,689,041), biopolymers (see, e.g., U.S. Pat. No. RE37,543; U.S. Pat.Nos. 6,228,623; 5,958,745 and U.S. Publication No. US20030028917) andbiofuel production (see, e.g., U.S. Pat. No. 5,998,700). The geneticelements, methods, and transgenes described in the patents and publishedapplications listed above are incorporated herein by reference.

Alternatively, a transcribable polynucleotide molecule can influence anabove mentioned (or other) plant characteristic or phenotypes byencoding an antisense or RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense, inhibitory RNA (RNAi), or cosuppression-mediated mechanisms.The RNA could also be a catalytic RNA molecule (a ribozyme) engineeredto cleave a desired endogenous mRNA product. Thus, any transcribablepolynucleotide molecule that encodes a transcribed RNA molecule thataffects a phenotype, biochemical or morphological change of interest maybenefit from the transcriptional enhancement enabled by the sequencesand constructs provided herein.

The described SCBV enhancer or chimeric transcription regulatory regioncomprising one or more copies thereof can be incorporated into aconstruct with one or more marker genes (any transcribablepolynucleotide molecule whose expression can be screened for or scoredin some way) and tested in transient or stable plant analyses to providean indication of the regulatory element's gene expression pattern instable transgenic plants. Marker genes for use in the practice of suchembodiments include, but are not limited to transcribable polynucleotidemolecules encoding β-glucuronidase (GUS described in U.S. Pat. No.5,599,670) and green fluorescent protein (GFP described in U.S. Pat.Nos. 5,491,084 and 6,146,826), proteins that confer antibioticresistance, or proteins that confer herbicide tolerance. Usefulantibiotic resistance markers, including those encoding proteinsconferring resistance to kanamycin (nptII), hygromycin B (aph IV),streptomycin or spectinomycin (aad, spec/strep) and gentamycin (aac3 andaacC4) are known in the art. Herbicides for which transgenic planttolerance has been demonstrated and the method of the present inventioncan be applied, include but are not limited to: glyphosate, glufosinate,sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione,protoporphyrionogen oxidase inhibitors, and isoxasflutole herbicides.Polynucleotide molecules encoding proteins involved in herbicidetolerance are known in the art, and include, but are not limited to apolynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS described in U.S. Pat. Nos. 5,627,061, 5,633,435,6,040,497 and in U.S. Pat. No. 5,094,945 for glyphosate tolerance);polynucleotides encoding a glyphosate oxidoreductase and aglyphosate-N-acetyl transferase (GOX described in U.S. Pat. No.5,463,175 and GAT described in U.S. publication No. 20030083480); apolynucleotide molecule encoding bromoxynil nitrilase (Bxn described inU.S. Pat. No. 4,810,648 for Bromoxynil tolerance); a polynucleotidemolecule encoding phytoene desaturase (crtl) described in Misawa et al.(Plant J. 4:833-840, 1993) and Misawa et al. (Plant J. 6:481-489, 1994)for norflurazon tolerance; a polynucleotide molecule encodingacetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan etal. (Nucl. Acids Res. 18:2188-2193, 1990) for tolerance to sulfonylureaherbicides; a polynucleotide molecule encoding a dicamba-degradingoxygenase enzyme (described in U.S. Patent Publications US20030135879and US20030115626, for dicamba tolerance); and the bar gene described inDeBlock et al. (EMBO J. 6:2513-2519, 1987) for glufosinate and bialaphostolerance. The regulatory elements of the present disclosure can expresstranscribable polynucleotide molecules that encode phosphinothricinacetyltransferase, glyphosate resistant EPSPS, aminoglycosidephosphotransferase, hydroxyphenyl pyruvate dehydrogenase, hygromycinphosphotransferase, neomycin phosphotransferase, dalapon dehalogenase,bromoxynil resistant nitrilase, anthranilate synthase, glyphosateoxidoreductase and glyphosate-N-acetyl transferase.

Constructs containing at least one SCBV enhancer (for instance, in thecontext of a chimeric transcription regulatory region) operably linkedto a marker gene or other nucleotide sequence of interest may bedelivered to a tissues (e.g., transformed) and the tissues analyzed bythe appropriate mechanism, depending on the marker or sequence that isbeing transcribed. Such quantitative or qualitative analyses may be usedas tools to evaluate the potential expression profile of a regulatoryelement when operatively linked to a gene of agronomic interest instable plants. Marker gene can be used in a transient assay; methods oftesting for marker gene expression in transient assays are known tothose of ordinary skill in the art. Transient expression of marker geneshas been reported using a variety of plants, tissues, and DNA deliverysystems. For example, transient analyses systems include but are notlimited to direct gene delivery via electroporation or particlebombardment of tissues in any transient plant assay using any plantspecies of interest. Such transient systems would include but are notlimited to electroporation of protoplasts from a variety of tissuesources or particle bombardment of specific tissues of interest. Thepresent disclosure encompasses use of any transient expression system toevaluate regulatory elements operably linked to any transcribablepolynucleotide molecule, including but not limited to marker genes orgenes of agronomic interest. Examples of plant tissues envisioned totest in transients via an appropriate delivery system would include butare not limited to leaf base tissues, callus, cotyledons, roots,endosperm, embryos, floral tissue, pollen, and epidermal tissue.

VII. Plant Transformation

A plant transformation construct containing an enhancer element (ormultiple copies thereof) or a chimeric transcription regulatory regionsuch as is described herein may be introduced into plants using anyplant transformation method. Methods and materials for transformingplants by introducing a plant expression construct into a plant genomein the practice of this invention can include any of the well-known anddemonstrated methods including electroporation (e.g., U.S. Pat. No.5,384,253), microprojectile bombardment (e.g., U.S. Pat. Nos. 5,015,580;5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865),Agrobacterium-mediated transformation (e.g., U.S. Pat. Nos. 5,824,877;5,591,616; 5,981,840; and 6,384,301), and protoplast transformation(e.g., U.S. Pat. No. 5,508,184). It will be apparent to those of skillin the art that a number of transformation methodologies can be used andmodified for production of stable transgenic plants from any number oftarget crops of interest.

Specific methods for transforming dicots are known to those skilled inthe art. By way of example, transformation and plant regenerationmethods have been described for a number of crops including, but notlimited to, cotton (Gossypium hirsutum), soybean (Glycine max), peanut(Arachis hypogaea), and members of the genus Brassica.

Likewise, specific methods for transforming monocots are also known tothose skilled in the art. By way of example transformation and plantregeneration methods have been described for a number of cropsincluding, but not limited to, barley (Hordeum vulgarae); maize (Zeamays); oats (Avena sativa); orchard grass (Dactylis glomerata); rice(Oryza sativa, including indica and japonica varieties); sorghum(Sorghum bicolor); sugar cane (Saccharum sp); tall fescue (Festucaarundinacea); turfgrass species (e.g. Agrostis stolonifera, Poapratensis, Stenotaphrum secundatum); wheat (Triticum aestivum), andalfalfa (Medicago sativa).

The transformed plants may be analyzed for the presence of the gene(s)of interest and the expression level and/or profile conferred by thechimeric transcription regulatory regions described herein. Numerousmethods are available to those of ordinary skill in the art for theanalysis of transformed plants. For example, methods for plant analysisinclude Southern and northern blot analysis, PCR-based (or other nucleicacid amplification-based) approaches, biochemical analyses, phenotypicscreening methods, field evaluations, and immunodiagnostic assays (e.g.,for the detection, localization, and/or quantification of proteins).

Enhanced expression of genes using the described SCBV enhancer has beendemonstrated in maize, but the enhancer is expected to function in otherplant species, possibly including dicots as well as monocots. Theenhancer element with four copies of the SCBV upstream region providedthe highest level of expression of the combinations studied herein.Fewer or more copies of the upstream region, as well as, combinationswith enhancer elements from other sources could also provide advantagesfor modulating gene expression. The same activators, constructs andapproaches may be useful for other crop species for which genes may beidentified because genome sequence is available or in progress(including Sorghum (Sorghum bicolor), Wheat (Triticum aestivum), Barley(Hordeum vulgare), Foxtail millet (Setaria italica), Sugarcane(Saccharum officinarum), Miscanthus giganteus or for which ‘activatedgenes’ may be identified by future genome sequencing efforts or perhapschromosomal synteny (including Oats (Avena sativa), Rye (Secalecereale), Pearl millet (Pennisetum glaucum), Finger millet (Eluesinecoracana), Proso millet (Panicum miliaceum), Teff millet (Eragrostistef)), or for model grass species for which genomic sequence isavailable or in progress (including Purple False Brome (Brachypodiumdistachyon), Green bristlegrass (Setaria viridis)).

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

Example 1 Identification of Sequences Comprising Enhancer Element ofSugarcane Bacilliform Virus (SCBV) Promoter

This example demonstrates the identification of sequences including theSCBV promoter enhancer element.

A promoter fragment derived from the genome of a SCBV (GenBank AccessionNo. AJ277091, and described by Geijskes et al., Arch. Virol., 147:2393-2404, 2002) was first examined by transient expression assays todetermine which regions of the promoter sequence contain enhancerelement sequences. In the promoter analysis study, fragments derivedfrom the SCBV promoter (SEQ ID NO: 1) containing sequences from −839 to+106 bp (plasmid pSCBV839), from −576 to +106 bp (plasmid pSCBV576), andfrom −333 to +106 bp (plasmid pSCBV333) from the transcription startsite (defined as the +1 position) were cloned upstream of a codingregion for a firefly luciferase (LUC) reporter protein. Transcriptionwas terminated by a copy of the nopaline synthase (Nos) 3′ UTR region(as disclosed in bases 1847 to 2103 of GenBank Accession No. V00087.1,which is hereby incorporated by reference in its entirety; and FIG. 1).Transient transcriptional activities of these constructs were tested bytransforming them by particle bombardment into maize Hi-II suspensioncells (described in detail in Example 2 below) and monitoring activityof the LUC reporter gene. Luciferase activity was normalized in eachexperiment by co-transforming with a equimolar amount of the plasmid DNAcontaining an SCBV:LUC construct and DNA of a reference plasmidharboring a construct consisting of a maize ubiquitin 1 (ubi1) genepromoter (as disclosed in U.S. Pat. No. 5,510,474 which is herebyincorporated by reference in its entirety; essentially bases 7 to 1990of GenBank Accession No. S94464.1, which is hereby incorporated byreference in its entirety) driving expression of a GUS(beta-glucuronidase) coding region, and terminated by a maize PerS 3′UTR terminator (as disclosed in U.S. Pat. No. 6,699,984, which is herebyincorporated by reference in its entirety; e.g., construct ubil:GUS).Two days after bombardment, total protein was isolated from transformedcells and LUC enzymatic activity (expressed in Luciferase Units (LU)/mgprotein) and GUS enzymatic activity (expressed in GUS activity units(GU)/μg protein) were measured by methods found in, for example, (Maligaet al., Methods in Plant Molecular Biology. A Laboratory Course Manual.Cold Spring Harbor Laboratory Press, 1995). Relative activities of thetest promoters in the three SCBV:LUC constructs were compared bynormalizing LUC levels to GUS levels as the ratio of LU/mg protein:GU/μgprotein. The transient testing results showed that LUC activityincreased linearly with increasing concentrations of plasmid DNAbombarded, indicating that LUC activity is correlated with transcriptlevels. Further, the SCBV promoter fragment containing sequences from−576 bp upstream to +106 downstream of the transcription start site had66%±2% of the activity of the full-length promoter fragment (heredefined as containing the sequences from −839 bp upstream to +106downstream of the start site). In contrast, the promoter fragmentcontaining sequences from −333 bp upstream to +106 downstream of thetranscription start site had only 17%±1% of the activity of thefull-length promoter. Thus, sequences for most of the SCBV promoteractivity reside upstream of −333 bp from the transcription start site.

The portion of the SCBV promoter sequence capable of enhancingtranscription driven by a heterologous minimal promoter sequence wasexamined. As defined by these experiments, an enhancer element isoperationally identified as a short (200 to 300 bp) cis-acting DNAsequence, lacking a TATA-box, that, when placed 5′ proximal to aheterologous minimal promoter sequence, increases the expressionactivity of the heterologous minimal promoter in a reproducible andmeasurable fashion when tested in either a transient or stabletransformation system. Further, tandem duplications of the enhancerelement provide even higher levels of expression activity of theheterologous minimal promoter than do single copies of the enhancerelement. The heterologous minimal promoter element utilized in thisExample comprises bases from −100 to +106 of a maize alcoholdehydrogenase 1 (Adh1) gene promoter (corresponding to bases 997 to 1202of GenBank Accession No. X04049, which is hereby incorporated byreference in its entirety).

Two fragments derived from the SCBV promoter, comprising sequences from−503 to −222 bp and from −758 to −222 bp relative to the transcriptionstart site, were cloned 5′ to sequences comprising a minimal maize Adh1promoter fused to a coding region encoding a firefly luciferase (LUC)protein. Transcription of the chimeric genes was terminated by the Nos3′UTR as described above. Maize Hi-II suspension culture cells weretransformed by particle bombardment with DNAs of plasmids harboring LUCand GUS constructs, and enzymatic activities were measured and comparedas above. Plasmids containing the LUC constructs having the −503 to −222sequences or the −758 to −222 sequences placed 5′ to the minimal Adh1promoter showed 6-fold, and 4-fold, respectively, more LUC activityrelative to the minimal Adh1 promoter without the added SCBV sequences.Thus, sequences within these fragments of the SCBV promoter enhancetranscription activity mediated by a heterologous maize promoter.

The abilities of multiple copies of the −503 to −222 bP SCBV enhancerregion to increase expression mediated by the minimal Adh1 promoter wastested by cloning one, two or four copies of the −502 to −222 bpsequences 5′ to the minimal maize Adh1 promoter fused to the LUC codingregion (FIG. 3A). Plasmid DNAs harboring the constructs (as well asplasmid DNA having a reference ubil:GUS construct) were bombarded intomaize Hi-II suspension culture cells, and LUC and GUS activities weremeasured and compared as above. Cells bombarded with constructscontaining 1 copy, 2 copies, or 4 copies of the SCBV enhancer sequenceregion had more than 5 times, 6 times and 10 times, respectively, moreLUC activity than did cells bombarded with an analogous minimal Adh1promoter construct lacking SCBV enhancer sequences (FIG. 3B).

Nucleic acid bases comprising −502 to −222 bp of the SCBV promoter, asprovided in SEQ ID NO: 1, encode transcriptional activation activitythat can confer superior expression characteristics to a plant promoter.Further, transcriptional activation activity is increased by thestacking of multiple tandem copies of the bases comprising −502 to −222bp of the SCBV promoter, as provided in SEQ ID NO: 1. Further still, themethods and reagents provided herein may be further examined andutilized to provide even shorter sequences that retain transcriptionalactivation activity, or may be combined with other transcriptionalactivator elements and plant promoters in new combinations.

Example 2 Transient Expression Testing of SCBV:LUC and ub1:GUSConstructs in Maize Hi-II Suspension Culture Cells

This example describes transient expression testing of SCBV:LUC andub1:GUS constructs in maize Hi-II suspension culture cells.

Maize Hi-II suspension culture cells (Armstrong et al., Maize Genet.Coop. Newslett., 65:92-93, 1991) were transformed by particlebombardment with DNAs of plasmids harboring LUC and GUS constructsconstructed as described above, and enzymatic activities were measuredand compared. Bulk preparations of plasmid DNAs were prepared usingQiAfilter™ Plasmid Maxi Kits (Qiagen, Germantown, Md.) and quantity andquality were analyzed using standard molecular methods.

Preparation of Maize Hi-II Suspension Culture Cells for Bombardment.

The Hi-II cells were maintained on a shaker at 125 rpm in H9CP+ mediumat 28° in darkness (H9CP medium consists of MS salts 4.3 gm/L, sucrose3%, Casamino acids 200 mg/L, myo-inositol 100 mg/L, 2.4-D 2 mg/L, NAA 2mg/L, 1000×MS vitamins 1 mL/L, L-proline 700 mg/L, and coconut water(Sigma Aldrich, St. Louis, Mo.) 62.5 mL/L, pH 6.0). Prior tobombardment, the 2-day old Hi-II cultures were transferred to G-N6medium (CHU N6 medium 3.98 g/L, CHU N6 vitamins 1 mL/L (both CHUcomponents from PhytoTechnology Laboratories®, Lenexa, Kans.),Myo-inositol 100 mg/L, 2,4-D 2 mg/L and Sucrose 3%, pH 6.0) and allowedto grow for 24 hours. On the day of bombardment, the G-N6 grown cells(2.5 gm of cells) were transferred to sterile Whatman No. 1 filter disks(55 mm) placed on G-N6 medium containing 0.5 M D-sorbitol and 0.5 MD-mannitol and incubated for 4 hours. The osmotically adjusted cells areused for bombardment.

Preparation of Gold Particles with Plasmid DNAs and Bombardment Assay.

Gold particles (1 μm diameter, BioRad, Hercules, Calif.) were washedwith 70% ethanol for 10 minutes, then three times with sterile water.The particles were dispensed in 50% glycerol at a concentration of 120mg/mL. For a typical experiment, 150 μL (18 mg) of gold particles,approximately 5 μg of plasmid DNA, 150 μL of 2.5 M CaCl₂ and 30 μL 0.2 Mspermidine were combined. The reaction (total volume 375 μL) wasincubated at room temperature for 10 minutes with occasional gentlevortexing. The DNA coated-gold particles were briefly centrifuged,washed with 420 μL of 70% ethanol and then with 420 μL of 100% ethanol.The final pellet was resuspended in 110 μL of 100% ethanol and subjectedto a brief sonication (three bursts of 3 seconds each, with 1 minutebetween bursts) with a Branson 1450 sonicator. Aliquots of 12.2 μL ofthe gold-particles coated with DNA were spread on each of ninemacrocarriers (BioRad, Hercules, Calif.) and used in bombardment assaysusing a BioRad PDS1000/He system. The suspension culture cells weretransformed at a target distance of 9 cm using 3510 psi disks and eachplate was bombarded 3 times. Following bombardment, the cells wereincubated in the dark at 28° C., first for 12 hours on G-N6 containingD-sorbitol and D-mannitol medium, then on G-N6 plates for an additional36 hours. Cells were collected from the plates, blotted to remove bufferand extracted with 300 μL of 2×CCLT LUC extraction buffer (PromegaCorporation, Madison, Wis.). After centrifugation, about 600 μL ofprotein extract was collected. Protein concentrations were estimatedusing the Bradford assay.

LUC enzymatic activity (expressed in Luciferase Units (LU)/mg protein)and GUS enzymatic activity (expressed in GUS activity units (GU)/μgprotein) were measured by methods found in, for example, Maliga et al.(Methods in Plant Molecular Biology. A Laboratory Course Manual. ColdSpring Harbor Laboratory Press, 1995). Relative activities of the testpromoters in SCBV:LUC constructs were compared by normalizing LUC levelsto GUS levels as the ratio of LUC/mg protein:GUS/μg protein.

Example 3 Plasmids for Activation Tagging in Maize Plants

This example describes generation of Agrobacterium superbinary plasmids.

The superbinary system is a specialized example of an Agrobacteriumshuttle vector/homologous recombination system (Komari et al., Meth.Mol. Biol. 343:15-41, 2006, Komari et al., Plant Physiol. 114:1155-1160,2007; see also European Patent No. EP604662B1 and U.S. Pat. No.7,060,876 each of which is incorporated by reference in its entirety).The Agrobacterium tumefaciens host strain employed with the superbinarysystem is LBA4404(pSB1). Strain LBA4404(pSB1) harbors twoindependently-replicating plasmids, pAL4404 and pSB1. pAL4404 is aTi-plasmid-derived helper plasmid which contains an intact set of virgenes (from Ti plasmid pTiACH5), but which has no T-DNA region (and thusno T-DNA left and right border repeat sequences). Plasmid pSB1 suppliesan additional partial set of vir genes derived from pTiBo542. Oneexample of a shuttle vector used in the superbinary system is pSB11,which contains a cloning polylinker that serves as an introduction sitefor genes destined for plant cell transformation, flanked by right andleft T-DNA border repeat regions. Shuttle vector pSB11 is not capable ofindependent replication in Agrobacterium, but is stably maintainedtherein as a co-integrant plasmid when integrated into pSB1 by means ofhomologous recombination between common sequences present on pSB1 andpSB11. Thus, the fully modified T-DNA region introduced intoLBA4404(pSB1) on a modified pSB11 vector is productively acted upon andtransferred into plant cells by Vir proteins derived from two differentAgrobacterium Ti plasmid sources (pTiACH5 and pTiBo542). The superbinarysystem has proven to be particularly useful in transformation of monocotplant species (See Hiei et al., Plant J. 6:271-282, 1994, and Ishida etal., Nat. Biotechnol. 14:745-750, 1996).

A transformation plasmid for production of activation tagged maizeplants can include a cointegrant plasmid formed by homologousrecombination between the superbinary plasmid pSB1 and pEPP1088, havinga pSB11 vector backbone (see European Patent No. EP604662B1 and U.S.Pat. No. 7,060,876 each of which are hereby incorporated by reference).The cointegrant plasmid is referred to as pSB1::pEPP1088 or as a ZeaTAGvector. The structure of pEPP1088 was validated by restriction enzymeanalysis and DNA sequence determination of selected regions of theconstruct. pEPP1088 contains, positioned between Left (LB) and Right(RB) T-DNA border sequences provided by the pSB11 plasmid, 4 copies ofthe −502 to −222 bp SCBV enhancer sequences described above and aselectable marker gene comprised of a rice (Oryza sativa) actin genepromoter with associated intron 1 and 5′ UTR (essentially as disclosedas bases 12 to 1411 of GenBank Accession No. EU155408.1 which is herebyincorporated by reference in its entirety), a coding sequence for anAAD-1 herbicide tolerance protein as disclosed in U.S. PatentApplication No. 20090093366, and a 3′ UTR terminator sequence from maizelipase gene essentially as disclosed as bases 921 to 1277 of GenBankAccession No. gbIL35913.11MZELIPASE and in U.S. Pat. No. 7,179,902 eachof which is hereby incorporated by reference in its entirety.

The T-DNA of pEPP1088 (and as present in pSB1::pEPP1088) integrates atrandom locations in maize chromosomes when introduced into maize cellsby Agrobacterium mediated transformation. Selection for transformedmaize cells is provided by the constitutively expressed AAD1 selectablemarker gene in the T-DNA. The T-DNA carrying tandem copies of the potent−502 to −222 bp SCBV transcriptional enhancer activator element causesaberrant expression of native genes nearby the integration site,thereby, in some instances, providing new identifiable traits to plantsregenerated from the transformed tissues. Modern molecular biologymethods are available which facilitate the isolation and identificationof the affected genes near the acceptor site, thus providing theisolated genes for further exploitation.

Example 4 Agrobacterium-Mediated Transformation of Maize

This example describes generation of Agrobacterium-mediatedtransformation of maize

Immature Embryo Production.

Seeds from a B104 inbred line were planted into 4-gallon-pots containingSunshine Custom Blend® 160 (Sun Gro Horticulture, Bellevue, Wash.). Theplants were grown in a greenhouse using a combination of high pressuresodium and metal halide lamps with a 16:8 hour Light:Dark photoperiod.To obtain immature embryos for transformation, controlledsib-pollinations were performed. Immature embryos were isolated at 10 to13 days post-pollination when embryos were approximately 1.4 to 2.0 mmin size.

Infection and Co-Cultivation.

Maize ears were surface sterilized by immersing in 50% commercial bleachwith Tween 20 (1 or 2 drops per 500 mL) for 10 minutes and triple-rinsedwith sterile water. A suspension of Agrobacterium cells containing asuperbinary vector cointegrant plasmid was prepared by transferring 1 or2 loops of bacteria grown on YEP solid medium containing 50 mg/LSpectinomycin, 10 mg/L Rifampicin, and 50 mg/L Streptomycin at 28° C.for 3 days or 25° C. for 4 days into 5 mL of liquid infection medium (MSsalts, ISU Modified MS Vitamins, 3.3 mg/L Dicamba, 68.4 gm/L sucrose, 36gm/L glucose, 700 mg/L L-proline, pH 5.2) containing 100 μMacetosyringone. The solution was gently pipetted up and down using asterile 5 mL pipette until a uniform suspension was achieved, and theconcentration was adjusted to an optical density of 0.3 to 0.5 at 600 nm(OD₆₀₀) using an Ultrospec 10 Cell Density Meter (GE Healthcare/AmershamBiosciences, Piscataway, N.J.). Immature embryos were isolated directlyinto a micro centrifuge tube containing 2 mL of the infection medium.The medium was removed and replaced twice with 1 to 2 mL of freshinfection medium, then removed and replaced with 1.5 mL of theAgrobacterium solution. The Agrobacterium and embryo solution wasincubated for 5 minutes at room temperature and then transferred toco-cultivation medium which contained MS salts, ISU Modified MSVitamins, 3.3 mg/L Dicamba, 30 gm/L sucrose, 700 mg/L L-proline, 100mg/L myo-inositol, 100 mg/L Casein Enzymatic Hydrolysate, 15 mg/L AgNO₃,100 μM acetosyringone, and 2.3 to 3 gm/L Gelzan™ (Sigma-Aldrich, St.Louis, Mo.), at pH 5.8. Co-cultivation incubation was for 3 to 4 days at25° C. under either dark or 24-hour white fluorescent light conditions(approximately 50 μEm⁻²s⁻¹).

Resting and Selection.

After co-cultivation, the embryos were transferred to a non-selectionMS-based resting medium containing MS salts, ISU Modified MS Vitamins,3.3 mg/L Dicamba, 30 gm/L sucrose, 700 mg/L L-proline, 100 mg/Lmyo-inositol, 100 mg/L Casein Enzymatic Hydrolysate, 15 mg/L AgNO₃, 0.5gm/L MES (2-(N-morpholino)ethanesulfonic acid monohydrate;PhytoTechnologies Labr., Lenexa, Kans.), 250 mg/L Carbenicillin, and 2.3gm/L Gelzan™, at pH 5.8. Incubation was continued for 7 days at 28° C.under either dark or 24-hour white fluorescent light conditions(approximately 50 μEm⁻²s⁻¹). Following the 7 day resting period, theembryos were transferred to selective medium. For selection of maizetissues transformed with a superbinary plasmid containing a plantexpressible AAD1 selectable marker gene, the MS-based resting medium(above) was used supplemented with Haloxyfop. The embryos were firsttransferred to selection media containing 100 nM Haloxyfop and incubatedfor 1 to 2 weeks, and then transferred to 500 nM Haloxyfop and incubatedfor an additional 2 to 4 weeks. Transformed isolates were obtained overthe course of approximately 5 to 8 weeks at 28° C. under either dark or24-hour white fluorescent light conditions (approximately 50 μEm⁻²s⁻¹).Recovered isolates were bulked up by transferring to fresh selectionmedium at 1 to 2 week intervals for regeneration and further analysis.

Those skilled in the art of maize transformation will understand thatother methods of selection of transformed plants are available whenother plant expressible selectable marker genes (e.g., herbicidetolerance genes) are used.

Pre-Regeneration.

Following the selection process, cultures exposed to the 24-hour lightregime were transferred to an MS-based pre-regeneration mediumcontaining MS salts, ISU Modified MS Vitamins, 45 gm/L sucrose, 350 mg/LL-proline, 100 mg/L myo-inositol, 50 mg/L Casein Enzymatic Hydrolysate,1 mg/L AgNO₃, 0.25 gm/L MES, 0.5 mg/L naphthaleneacetic acid, 2.5 mg/Labscisic acid, 1 mg/L 6-benzylaminopurine, 250 mg/L Carbenicillin, 2.5gm/L Gelzan™, and 500 nM Haloxyfop, at pH 5.8. Incubation was continuedfor 7 days at 28° under 24-hour white fluorescent light conditions(approximately 50 μEm⁻²s⁻¹).

Regeneration and Plantlet Isolation.

For regeneration, the cultures were transferred to an MS-based primaryregeneration medium containing MS salts, ISU Modified MS Vitamins, 60gm/L sucrose, 100 mg/L myo-inositol, 125 mg/L Carbenicillin, 2.5 gm/LGelzan™, and 500 nM Haloxyfop, at pH 5.8. After 2 weeks at 28° undereither dark or 24-hour white fluorescent light conditions (approximately50 μEm⁻²s⁻¹), tissues were transferred to an MS-based secondaryregeneration medium composed of MS salts, ISU Modified MS Vitamins, 30gm/L sucrose, 100 mg/L myo-inositol, 3 gm/L Gelzan™, at pH 5.8, with, orwithout, 500 nM Haloxyfop. Regeneration/selection was continued for 2weeks at 28° under either 16-hour or 24-hour white fluorescent lightconditions (approximately 50 μEm⁻²s⁻¹). When plantlets reached 3 to 5 cmin length, they were excised and transferred to secondary regenerationmedium (as above, but without Haloxyfop) and incubated at 25° under16-hour white fluorescent light conditions (approximately 50 μEm⁻²s⁻¹)to allow for further growth and development of the shoot and roots.

Seed Production.

Plants were transplanted into Metro-Mix® 360 soilless growing medium(Sun Gro Horticulture) and hardened-off in a growth room. Plants werethen transplanted into Sunshine Custom Blend 160 soil mixture and grownto flowering in the greenhouse. Controlled pollinations for seedproduction were conducted.

Example 5 SCBV Enhancer Activity in Stably Transformed Maize Cells

Genomic DNA was isolated (Qiagen DNeasy® Plant Mini Kit; Qiagen,Germantown, Md.) from ten To plants regenerated from transformed B104immature embryos, and the genomic locations of the integrated T-DNAstransferred from pSB1::pEPP1088 were determined by inverse PCR cloningand DNA sequencing of the inverse PCR amplified products. The identitiesof genes represented by the flanking coding regions positioned within 10kb of the 4×SCBV enhancer were determined by BLAST searches (Altschul etal., J. Mol. Biol., 215: 403-410, and Karlin et al., Proc. Natl. Acad.Sci. USA 87: 2264-2268, 1990) using the flanking sequences as querysequences. Analyses of the BLAST results revealed that the T-DNAs, andhence the 4×SCBV enhancers, were integrated at a different genomiclocation in each of the 10 lines, and therefore the 4×SCBV enhancers areflanked by different genes in each line (Table 1).

Total RNA was isolated (Qiagen RNeasy® Plant Mini Kit, Qiagen,Germantown, Md.) from leaf tissues of the ten T₀ lines. Transcriptaccumulation of the identified flanking genes was compared between theappropriate To plants and non-transformed control plants by reversetranscription and RT-PCR (Real Time PCR), using primers specific for therelevant genes flanking the 4×SCBV enhancers. As a control, transcriptaccumulation for the endogenous GAPDH gene was also determined.

RT-PCR products revealed increased accumulation of transcriptsoriginating from 3 of the different flanking genes in these lines. The4×SCBV enhancers are located 2.6 kb and 2.8 kb upstream of the affectedflanking genes in 2 of the T₀ lines, and 478 bp downstream of theaffected flanking gene in the third To line. Thus, these resultsindicate that the 4×SCBV enhancers delivered by T-DNA causestrand-independent increased accumulation of transcripts of genes nearbythe integration site. Table 1 indicates the flanking genes identifiedand the results of analyses of their transcription levels.

TABLE 1 Effect 4XSCBV enhancer on the RNA accumulation of the flankinggenes in 10 T0 plants. Distance to the RNA T0 Plant ID 4XSCBV (bp)Flanking Gene Name Accumulation ZT00031845 1197 P-loop containing NTPhydrolases No change ZT00032132 5′-UTR A protein that helps vesicularfusion No change proteins ZT00036435 2644 DEAD-box-like helicaseIncreased ZT00034545 1972 High mobility group-like nuclear protein Nochange ZT00036729 EST Unknown protein No change ZT00035749 2818 Unknownprotein (GRMZM2G115661) Increased ZT00033904 830 Unknown protein Nochange ZT00036426 79 Ribosomal protein L22/L17; T0 plant is No changetall ZT00036426 2150 Signal peptide No change ZT00035050 478 from the3′-end Unknown gene (GRMZM2G139336) Increased

One skilled in the fields of maize genetics and plant molecular biologywill realize that, depending upon the nature of the affected genes, theincreased expression of adjacent genes induced by 4×SCBV enhancers willin some cases confer upon the transgenic plant new and valuable traits.Collectively, plants having the 4×SCBV enhancers represent aZeaTAG-marked population. The traits may be the result of increasedaccumulation of the affected gene's encoded protein per se, as forexample, increased accumulation of a nutritionally desirable protein inthe seed, or the result of a downstream effect whereby the gene productof the immediately affected gene controls the expression of one or amultitude of other genes (as in the case of, for example,transcriptional activator/repressor genes). The random nature ofintegration location of introduced T-DNAs, coupled with standard plantbreeding methods, may be used to establish large populations of plantscomprising a library of T-DNA bearing plants having activator elementspositioned within an effective distance of all or most genes within themaize genome, and thus provides the opportunity for all or most maizegenes to be transcriptionally activated.

Plant-level screening for phenotypes of economic importance is possibleunder growth chamber, greenhouse, or field environments. As shown here,molecular biology methods such as inverse PCR enable the isolation of anintegrated T-DNA and substantial lengths of genomic DNA flanking theintegrated T-DNA from plants exhibiting a desirable phenotype. Further,methods such as genome walking techniques allow the determination ofeven more extensive regions of genomic DNA sequence, thus enablingidentification of the genes present in proximity to introduced activatorelements. High throughput methods such as microarray analysis and moregene specific analytical methods enable identification andquantification of affected transcript levels. Candidate genes involvedin relevant agronomic traits may thus identified, isolated, and furthercharacterized and exploited to provide new and valuable varieties ofcrops.

Conversely, the new trait may be the result of disruption of maize genefunction due to the integration of the T-DNA having the 4×SCBV enhancersinto the coding region or expression regulatory regions of the maizegene. If such is the case, the T-DNA having the 4×SCBV enhancers andsurrounding genomic regions can be isolated and further characterized.

Example 6 Forward Genetic Screening of the ZeaTAG Population

This example describes forward genetic screening of the ZeaTAGpopulation for altered phenotypes.

Drought Stress Screens

To identify ZeaTAG lines that contain mutations conferring droughttolerance, plants from individual ZeaTAG events are planted in a field.Water is withheld to cause drought stress during the reproductive phaseof the growth cycle; roughly 2 weeks prior to flowering to approximately2 weeks after flowering. The target is to achieve 4 weeks of stressperiod at flowering stage. Environmental modeling is used to predictaccurate corn evapotranspiration demand based on soil moisturemonitoring and weather data (air temperature, vapor pressure deficit,wind speed, and net radiation). Plants are monitored for droughtsymptoms such as leaf rolling by visual observation, increased leaftemperature by infrared thermometers, reduced photosynthesis bychlorophyll fluorescence and reduced yield by measuring grainproduction. Plants that show significantly less leaf rolling, lower leaftemperature, higher rates of photosynthesis or have significantly moreyield under water stress conditions are identified and used insubsequent screens.

ZeaTAG events displaying significantly more drought tolerance areplanted in a replicated field trial to confirm the drought tolerantphenotype. These events are planted in a randomize split block designwith at least 3 replications. One block is irrigated with watersufficient to prevent water stress. The other block is grown under waterdeficient conditions as described above. Plants are monitored for leafrolling, increased leaf temperature, decreased photosynthesis anddecreased yield as described above. Plants with significantly less leafrolling, lower leaf temperature, greater photosynthesis or greater yieldthan untransformed control plants are considered to have passed thesecondary screen.

Nitrogen Use Efficiency Screens

To identify ZeaTAG events with greater nitrogen use efficiency thannon-transgenic control plants a primary screen is performed. Plantscontaining approximately 40,000 ZeaTAG containing events are grown inthe field under nitrogen deficient conditions. Plants are grown infields with less than 35 lbs of N per acre. Plants are monitored forchlorosis by visual inspection, increased leaf temperature by infraredthermometers, and decreased yield by grain harvest. These parameters arecompared with non-transgenic control plants. ZeaTAG lines showing lesschlorosis, lower leaf temperature, higher photosynthetic rates orgreater yields than non-transgenic control lines are evaluated insecondary screens.

As a secondary screen, ZeaTAG events displaying significantly morenitrogen use efficiency are planted in a replicated field trial toconfirm the phenotype. These events are planted in a randomize splitblock design with at least 3 replications. One block is irrigated withsufficient nitrogen fertilizer to prevent nitrogen stress. The otherblock is grown under nitrogen deficient conditions as described above.Plants are monitored for chlorosis by visual inspection, increased leaftemperature by infrared thermometers, and decreased yield by grainharvest. Plants with significantly less chlorosis, lower leaftemperature, greater photosynthesis or greater yield than untransformedcontrol plants are considered to have passed the secondary screen.

Once the phenotype has been confirmed in the secondary screen, thephenotype is tested for genetic linkage with the ZeaTAG insertion byscreening the progeny of a cross between the non-transformed parentalline and the ZeaTAG line. When plants containing the ZeaTAG elementdisplay the phenotype and plants that do not contain the ZeaTAG elementdo not, the phenotype is considered to be genetically linked with theinsert and likely to be caused by the ZeaTAG element. To identify geneswhose expression may be affected by the ZeaTAG element, the location ofthe ZeaTAG element within the genome is determined.

The genomic location of the ZeaTAG element is determined by isolatinggenomic sequences flanking the ZeaTAG element and comparing thesesequences to the genomic sequence of maize. Sequences flanking theZeaTAG element can be determined by a number of molecular biologicaltechniques, including but not limited to, inverse PCR (iPCR) (Ochman etal., Genetics, 120: 621-6231988), TAIL (Liu et al., Plant Journal 8:457-463, 1995) and ligation-mediated PCR (LMPCR) Prod'hom et al., FEMSMicrobiol Lett. 158: 75-81, 1998). These sequences are compared togenomic sequences by sequence alignment tools such as BLAST to identifythe location of the ZeaTAG element within the genome.

Genes flaking or interrupted by the ZeaTAG element are determined byexamining the annotated genome. Transcription of genes flanking theZeaTAG element may be responsible for the mutant phenotype. These genesmay be over-expressed in wild-type maize to test whether they can confera similar phenotype. To test this, the genes are cloned intotransformation vectors driven by strong promoters or by their ownpromoter with enhancer sequences flanking them to enhance transcription.These vectors are introduced into wild-type maize by transformation andplants resulting from this transformation are tested for the phenotype.

Similarly, genes interrupted by the ZeaTAG element may cause thephenotype. To confirm that a gene interrupted by the element isresponsible for the phenotype, expression of the gene can be disruptedand plants containing this disruption can be tested for the phenotype.The disruption of expression of specific genes can be accomplished by anumber of methods know to those skilled in the art including but notlimited to antisense RNA, artificial micro RNAs and identifyingmutations in the gene by TILLING.

Example 7 Reverse Genetic Screening of the ZeaTAG Population

This example describes reverse genetic screening of the ZeaTAGpopulation for mutations.

Reverse genetic screening is looking for mutations affecting specificgenes and subsequently testing the identified line for a mutantphenotype. The ZeaTAG population can be used in reverse genetic analysesin several ways including but not limited to generating a collectionFlanking Sequence Tags for the population (Jeong et al., The PlantJournal 45: 123-132, 2006) and generating an indexed collection ofpooled samples of DNA from the ZeaTAG population (May et al., MolecularBiotechnology 20: 209-221, 2002).

A collection of Flanking Sequence Tags is generated by sampling leaftissue from the ZeaTAG population, isolating DNA from each,identification of sequences flanking the insert and storing thesequences in a searchable database where the sequences are linked to theevents from which they came. Genomic DNA is isolated using the QiagenDNAeasy Plant Kit (Qiagen, Germantown, Md.) using the protocolrecommended by the manufacturer. Sequences flanking the insert areidentified using Ligation Mediated PCR (Mueller et al., Science 246:780-786, 1989) as modified by Yephremov and Saedler (Plant Journal 21:295-305, 2000). Briefly, genomic DNA from a ZeaTAG line is fragmentedrestriction enzyme digestion and denatured. A biotinylatedoligonucleotide primer complementary to the sequence at the end of theZeaTAG element is hybridized to the fragmented DNA and extended by DNApolymerase. Streptavidin coated magnetic beads are added to the mixtureto bind DNA fragments containing DNA fragments extended from thisprimer. A double-stranded DNA adaptor of known sequence is ligated tothe unknown end. These fragments are PCR amplified usingoligonucleotides complementary to sequences within the ZeaTAG elementand the DNA adaptor at the other end. The sequence of the PCR fragmentis then determined and mapped to the maize genomic sequence by BLAST.These sequences locate the site of insertion of the ZeaTAG element.Genes within a ˜10 kbp may be up-regulated by the enhancer sequenceswithin the ZeaTAG element.

Plants containing insertions in or near genes that are hypothesized tocause a phenotype can be identified by searching the database. Plantscontaining these events can be tested for the phenotype.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A chimeric transcription regulatory region comprising: oneor more copies of the sugarcane bacilliform viral (SCBV) enhancerelement shown in position 337 to position 618 of SEQ ID NO: 1, or ahomolog thereof; and operably linked thereto, a promoter comprising anRNA polymerase binding site and a mRNA initiation site, wherein when anucleotide sequence of interest is transcribed under regulatory controlof the chimeric transcription regulatory region, the amount oftranscription product is enhanced compared to the amount oftranscription product obtained with the chimeric transcriptionregulatory region comprising the promoter and not comprising the SCBVenhancer sequence(s).
 2. The chimeric transcription regulatory region ofclaim 1, wherein the promoter is obtained from the upstream region of aplant virus gene, a bacterial gene, a fungal gene, a plant nuclear gene,a plant extra-nuclear gene, an invertebrate gene, or a vertebrate gene.3. A construct comprising the transcriptional initiation region of claim1 operably linked to a transcribable polynucleotide molecule operablylinked to a 3′ transcription termination polynucleotide molecule.
 4. Theconstruct of claim 3, wherein said transcribable polynucleotide moleculeconfers an agronomic trait to a plant in which it is expressed.
 5. Atransgenic plant stably transformed with the construct of claim
 3. 6.The transgenic plant of claim 5, wherein the transcribablepolynucleotide molecule confers an agronomic trait to a plant in whichit is expressed.
 7. A seed of the transgenic plant of claim 5, whereinthe seed comprises the construct.
 8. The transgenic plant of claim 5,which plant is a maize plant
 9. A transgenic plant cell comprising thechimeric transcription regulatory region of claim
 1. 10. A method ofproducing a transgenic plant comprising transforming a plant cell ortissue with the construct of claim
 3. 11. The method of claim 10,wherein the transgenic plant is a dicotyledon.
 12. The method of claim10, wherein the transgenic plant is a monocotyledon.
 13. A plant cell ortissue transformed with the construct of claim
 3. 14. The plant cell ortissue of claim 13, wherein the plant cell or tissue is from adicotyledon.
 15. The plant cell or tissue of claim 13, wherein the plantcell or tissue is derived from a monocotyledon.
 16. A plant cell, fruit,leaf, root, shoot, flower, seed, cutting and other reproductive materialuseful in sexual or asexual propagation, progeny plants inclusive of F1hybrids, male-sterile plants and all other plants and plant productsderivable from the transgenic plant of claim
 5. 17. A maize plant cell,tissue or plant comprising one or more copies of the sugarcanebacilliform viral (SCBV) enhancer element shown in position 337 toposition 618 of SEQ ID NO: 1, or a homolog thereof, in which the one ormore copies of the SCBV enhancer element is inserted into a genome ofthe maize plant cell, tissue or plant at a random location.
 18. Themaize plant cell, tissue or plant of claim 17, wherein the SCBV enhancerimparts enhanced transcription of a nucleotide sequence of interestwhich is under regulatory control of the SCBV enhancer as compared totranscription of the nucleotide sequence of interest in the absence ofthe SCBV enhancer.